N,N′-diarylurea compounds and N,N′-diarylthiourea compounds as inhibitors of translation initiation

ABSTRACT

Compositions and methods for inhibiting translation initiation are provided. Compositions, methods and kits for treating (1) cellular proliferative disorders, (2) non-proliferative, degenerative disorders, (3) viral infections, and/or (4) disorders associated with viral infections, using N,N′-diarylureas and/or N,N′-diarylthiourea compounds are described.

RELATED APPLICATION DATA

This application is a continuation application which claims priority toU.S. patent application Ser. No. 13/322,607, filed on Jan. 24, 2012,which is a National Stage Application under 35 U.S.C. 371 of co-pendingPCT application PCT/US2010/036584 designating the United States andfiled May 28, 2010; which claims the benefit of U.S. provisionalapplication Ser. No. 61/181,920 filed May 28, 2009, each of which arehereby incorporated by reference in their entireties.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under 5 U19 CA87427awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD

The present invention relates to novel compounds which inhibittranslation initiation, pharmaceutical compositions of the novelcompounds, and methods of treating medical disorders.

BACKGROUND

Translation, the mRNA-directed synthesis of proteins, occurs in threedistinct steps: initiation, elongation and termination. Translationinitiation is a complex process in which the two ribosomal subunits andmethionyl tRNA (Met-tRNA_(i)) assemble on a properly aligned mRNA tocommence chain elongation at the AUG initiation codon. The establishedscanning mechanism for initiation involves the formation of a ternarycomplex among eukaryotic initiation factor 2 (eIF2), GTP andMet-tRNA_(i). The ternary complex recruits the 40S ribosomal subunit toform the 43S pre-initiation complex. This complex recruits mRNA incooperation with other initiation factors such as eukaryotic initiationfactor 4E (eIF4E), which recognizes the 7-methyl-guanidine cap (m-⁷GTPcap) in an mRNA molecule and forms the 48S pre-initiation complex. Caprecognition facilitates the 43S complex entry at the 5′ end of a cappedmRNA. Subsequently, this complex migrates linearly until it reaches thefirst AUG codon, where a 60S ribosomal subunit joins the complex, andthe first peptide bond is formed (Pain (1996) Eur. J. Biochem.,236:747-771). After each initiation, the GTP in the ternary complex isconverted to GDP. The eIF2 GDP binary complex must be converted to eIF2GTP by the guanidine exchange factor, eIF2B for a new round oftranslation initiation to occur. Inhibition of this exchange reaction byphosphorylation of eIF2α reduces the abundance of the ternary complexand inhibits translation initiation. Forced expression ofnon-phosphorylatable eIF2α or Met-tRNA_(i) causes transformation ofnormal cells (Marshall (2008) Cell 133:78; Berns (2008) Cell 133:29). Incontrast, pharmacologic agents that restrict the amount ofeIF2-GTP-Met-tRNA_(i) ternary complex inhibit proliferation of cancercells in vitro and tumors in vivo (Aktas (1998) Proc. Natl. Acad. Sci.U.S.A. 95:8280), Palakurthi (2000) Cancer Res. 60:2919, Palakurthi(2001) Cancer Res. 61: 6213). These findings indicate that more potentand specific agents that reduce amount of ternary complex are potentanti-cancer agents.

Several features of the mRNA structure influence the efficiency of itstranslation. These include the m-⁷GTP cap, the primary sequencesurrounding the AUG codon and the length and secondary structure of the5′ untranslated region (5′ UTR). Indeed, a moderately long, unstructured5′ UTR with a low G and C base content seems to be optimal to ensurehigh translational efficiency. Surprisingly, sequence analysis of alarge number of vertebrate cDNAs has shown that although mosttranscripts have features that ensure translational fidelity, many donot appear to be designed for efficient translation (Kozak (1991) J.Cell. Biol., 115:887-903). Many vertebrate mRNAs contain 5′ UTRs thatare hundreds of nucleotides long with a remarkably high GC content,indicating that they are highly structured because G and C bases tend toform highly stable bonds. Because highly structured and stable 5′ UTRsare the major barrier to translation, mRNAs with stable secondarystructure in their 5′ UTR are translated inefficiently and theirtranslation is highly dependent on the activity of translationinitiation factors. mRNAs with complex, highly structured 5′ UTRsinclude a disproportionately high number of proto-oncogenes such as theG1 cyclins, transcription and growth factors, cytokines and othercritical regulatory proteins. In contrast, mRNAs that encode globins,albumins, histones and other housekeeping proteins rarely have highlystructured, GC-rich 5′ UTRs (Kozak (1994) Biochimie, 76; 815-21; Kozak(1999) Gene, 234:187-208). The fact that genes encoding for regulatorybut not for housekeeping proteins frequently produce transcripts withhighly structured 5′ UTRs indicates that extensive control of theexpression of regulatory genes occurs at the level of translation. Inother words, low efficiency of translation is a control mechanism whichmodulates the yield of proteins such as cyclins, mos, c-myc, VEGF, TNF,among others, that could be harmful if overproduced.

Translation initiation is a critical step in the regulation of cellgrowth because the expression of most oncogenes and cell growthregulatory proteins is translationally regulated. One approach toinhibiting translation initiation has recently been identified usingsmall molecule known as translation initiation inhibitors. Translationinitiation inhibitors such as clotrimazole (CLT) inhibit translationinitiation by sustained depletion of intracellular Ca²⁺ stores.Depletion of intracellular Ca²⁺ stores activates “interferon-inducible”“double-stranded RNA activated” protein kinase (PKR) whichphosphorylates and thereby inhibits the a subunit of eIF2. Since theactivity of eIF2 is required for translation initiation, its inhibitionby compounds such as CLT reduces the overall rate of protein synthesis.Because most cell regulatory proteins are encoded for by mRNAscontaining highly structured 5′ UTRs, they are poorly translated andtheir translation depends heavily on translation initiation factors suchas eIF2 and eIF4. Therefore, inhibition of translation initiationpreferentially affects the synthesis and expression of growth regulatoryproteins such as G1 cyclins. Sequential synthesis and expression of G1cyclins (D1, E and A) is necessary to drive the cell cycle beyond therestriction point in late G1. Thus, the decreased synthesis andexpression of G1 cyclins resulting from CLT-induced inhibition oftranslation initiation causes cell cycle arrest in G1 and inhibitscancer cell and tumor growth (Aktas et al. (1998) Proc. Natl. Acad. Sci.USA, 95:8280-8285, incorporated herein by reference in its entirety forall purposes).

Like CLT, the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA)depletes internal calcium stores, and exhibits anti-carcinogenicactivity. Unlike CLT, however, EPA is a ligand of peroxisomeproliferator-activated receptor gamma (PPARγ), a fatty acid-activatedtranscription factor. Although EPA and other ligands of PPARγ, such astroglitazone and ciglitazone, inhibit cell proliferation, they do so ina PPARγ-independent manner (Palakurthi et al. (2000) Cancer Research,60:2919; and Palakurthi et al. (2001) Cancer Research, 61:6213,incorporated herein by reference in their entirety for all purposes).

SUMMARY

Embodiments of the present invention are directed to compounds thatinhibit translation initiation, and the use of such compounds orcombination of compounds for treating (1) proliferative disorders, (2)non-proliferative, degenerative disorders, (3) viral infections, (4)disorders associated with viral infections, and/or (5) disorderscharacterized by unwanted protein synthesis or diseases for whichreducing protein synthesis is advantageous.

In at least certain examples, the compounds are of substituteddiarylureas, more particularly, substituted N,N′-diarylurea compounds.In other examples, the compounds are substituted thioureas, moreparticularly, substituted N,N′-diarylthiourea compounds. In certainexemplary embodiments, substituted N,N′-diarylurea and/or substitutedN,N′-diarylthiourea compounds include compounds comprising Formula I,Formula II, Formula III, Formula IV and/or compounds set forth in Tables1-6, FIGS. 1-12 and the Appendix.

In certain examples, the substituted N,N′-diarylurea and/or substitutedN,N′-diarylthiourea compounds described herein cause phosphorylation ofeIF2α. In other examples, substituted N,N′-diarylurea and/or substitutedN,N′-diarylthiourea compounds are effective to inhibit translationinitiation.

In accordance with a method aspect, a method of treating a proliferativedisorder by providing and/or administering a compound of Formula Iand/or Formula II and/or Formula III and/or Formula IV to a mammal,e.g., a human or a non-human (e.g., a non-human primate), is provided.In one example, the proliferative disorder is cancer. In accordance withother examples, a method of treating a viral infection by providingand/or administering a compound of Formula I and/or Formula II and/orFormula III and/or Formula IV to a mammal, e.g. a human or a non-humanmammal, is provided.

In accordance with an additional aspect, kits are provided for thetreatment of (1) proliferative disorders, (2) non-proliferative,degenerative disorders, (3) viral infections, (4) disorders associatedwith viral infections, and/or (5) disorders characterized by unwantedprotein synthesis or diseases for which reducing protein synthesis isadvantageous In one aspect, the kits comprise a compound of Formula Iand/or Formula II and/or Formula III and/or Formula IV, apharmaceutically acceptable carrier, and optionally, instructions foruse. The pharmaceutical composition can be administered to a humansubject or a non-human subject depending on the disorder to be treated.

It will be recognized by the person of ordinary skill in the art thatthe compounds, compositions, methods and kits disclosed herein providesignificant advantages over prior technology. Compounds, compositions,methods and kits can be designed or selected to relieve and/or alleviatesymptoms in a patient suffering from one or more disorders. These andother aspects and examples are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings.

FIGS. 1A-ID graphically depict ternary complex assays. (A) KLN cells.(B) CRL-2351 cells. (C) Real-time PCR of CHOP. (D) Quantification ofCHOP to beta-actin Western blot.

FIG. 2 graphically depicts quantification of phosphorylated eIF2αrelative to total eIF2α.

FIGS. 3A-3D graphically depict the ratios of firefly luciferase (F-luc):renilla luciferase (R-luc) for compounds 1527 (A), 1780 (B), 1781 (C)and KM94748 (D) in PC-3 cells transfected with ether wild-type (WT)eIF2α or non-phosphorylatable eIF2α-S51A mutant.

FIGS. 4A-4D graphically depict ATF-4 assays using substitutedN,N′-diarylureas (numbers of compounds correspond to structures inTable 1) in CRL-2351 cells.

FIGS. 5A-5D graphically depict ATF-4 assays using substitutedN,N′-diarylureas (numbers of compounds correspond to structures inTable 1) in KLN cells.

FIGS. 6A-6D depict the development and validation of theeIF2-GTP-Met-tRNA_(i) ternary complex assay. A) Firefly and renillaluciferase open reading frames (ORFs) were cloned into pBISA plasmid togenerate two mRNAs that differ only in their coding region (pBISA-DL,top). This vector was further modified by cloning 5′ untranslated region(UTR) of mouse ATF-4 gene in frame with the AUG start codon of fireflyluciferase ORF (pBISA-DL^((ATF-4)), bottom). B) pBISA-DL^((ATF-4))construct was stably transfected into KLN-tTA cells and responsivenessof these cells to thapsigargin and tunicamycin was evaluated by a DualLuciferase Assay (DLR assay). The firefly/renilla (F/R) ratio in vehicletreated cells was taken as 1. C) The second uORF in the ATF-4 5′ UTR wasfused in frame to AUG start codon of firefly luciferase to remove eIF2αphosphorylation dependent induction of ATF-4 translation, the plasmidwas transiently transfected into KLN-tTA cells. The cells were treatedwith DMSO (vehicle) Thapsigargin (100 nM) or tunicamycin (1 μg/ml) andR/F ratio was determined by DLR assay. D) StableKLN-tTA/pBISA-DL^((ATF-4)) cell line in B was plated into a 384 wellplate, half the plate was treated with TG and other half with thevehicle (DMSO). Firefly/renilla ratio was determined by DLR assay andplotted.

FIGS. 7A-7D depict the validation of N,N′-diarylureas as modifiers ofthe eIF2-GTP-Met-tRNA_(i) ternary complex. A) The structure of threeactive and one inactive N,N′-diarylurea compounds selected for furtherstudies. B) KLN-tTA/pBISA-DL^((ATF-4)) cells were incubated with theindicated concentrations of each N,N′-diarylurea compound andfirefly/renilla (F/R) ratio was determined by DLR assay. C)KLN-tTA/pBISA-DL^((ATF-4)) cells were incubated with the indicatedconcentrations of each N,N′-diarylurea compound and expression ofendogenous CHOP protein was determined by Western blot analysis. D)KLN-tTA/pBISA-DL^((ATF-4)) cells were incubated with 5 or 20 μM of eachN,N′-diarylurea compound and expression of endogenous CHOP mRNA wasdetermined by real time PCR analysis.

FIGS. 8A-8D depict that N,N′-diarylureas reduce the availability of theeIF2-GTP-Met-tRNA_(i) ternary complex in human cancer cells. A-C) PC-3,CR-L2351, and CRL-2813 human cancer cell lines were co-transfected withpBISA-DL^((ATF-4)) and ptTA plasmids. One day post-transfection, thecells were treated with the indicated concentrations ofN,N′-diarylureas. Firefly/renilla ratio was determined by DLR assay 8hours after treatment. D) PC-3, CR-L2351, and CRL-2813 human cancer celllines were treated with the indicated concentrations of N,N′-diarylureasfour 8 hours, and expression of endogenous CHOP mRNA was determined byreal-time PCR.

FIGS. 9A-9B depict that N,N′-diarylureas modify theeIF2-GTP-Met-tRNA_(i) ternary complex by causing the phosphorylation ofeIF2α. A) KLN-tTA/pBISA-DL^((ATF-4)) or PC-3 cell lines were incubatedwith N,N′-diarylureas, levels of total eIF2α was determined by Westernblot analysis with mouse monoclonal antibodies (Biosource International,MA) and the level of phosphorylated eIF2α was determined by Western blotanalysis with phosphor-serine 51 (Phos-eIF2α) specific recombinantrabbit monoclonal antibodies (Epitomics Inc, CA). B) The PC-3 cells inwhich endogenous eIF2α was replaced with recombinant WT ornon-phosphorylatable eIF2α-S51A mutant were co-transfected with tTA andpBISA-DL^((ATF-4)) dual luciferase expression vector and treated withindicated concentrations of N,N′-diarylurea compounds. Firefly/renillaratio was determined by DLR assay.

FIGS. 10A-10B depict that reducing availability of theeIF2-GTP-Met-tRNA_(i) ternary complex inhibits cancer cellproliferation. A) Various human and mouse cancer cell lines wereincubated with the indicated concentrations of N,N′-diarylureacompounds, net cell proliferation was determined by SRB assay. B) ThePC-3 human prostate cancer cells in which endogenous eIF2α was replacedwith recombinant WT eIF2α or non-phosphorylatable eIF2α-S51A mutant weretreated with the indicated concentrations of N,N′-diarylurea compoundsand cell proliferation was measured by Sulforhodamine B (SRB) assay.

FIGS. 11A-11B depict that reducing availability of theeIF2-GTP-Met-tRNA_(i) ternary complex modified expression of cell cycleregulatory proteins. A) Mouse KLN cells were incubated with 20 μM andhuman PC3 cancer cell lines were incubated with 5 μM N,N′-diarylureacompounds and expression of cyclin D1 or β-actin was determined byWestern blot analysis. B) KLN and PC-3 cells were treated as in A andexpression of cyclin D1 mRNA was determined with real time PCR.

FIGS. 12A-12D depict that the N,N′-Diarylurea compounds specificallyactivate HRI kinase. A) KLN-tTA/pBISA-DL^((ATF-4)) cells weretransfected with mock siRNA or siRNA targeting PKR, PERK, GCN2, HRI orPKR, PERK, GCN2 simultaneously. Cells were treated with compound #1781or DMSO and F-luc/R-luc ratio was determined by DLR. B)KLN-tTA/pBISA-DL^((ATF-4)) cells treated as in A, the expression of CHOPmRNA was determined by real-time PCR. C). KLN-tTA/pBISA-DL^((ATF-4))cells were transfected with mock siRNA or siRNA targeting HRI, the cellswere treated with N,N′-diarylurea compounds or vehicle and theF-luc/R-luc ratio was determined by DLR. D) Cells were transfected withmock siRNA or siRNA targeting PKR. PERK, GCN2, or HRI and knockdownefficiency for each gene was determined by real-time PCR.

FIGS. 13A-13E depict that the N,N′-diarlyurea compounds specificallyactivate HRI kinase. A) KLN-tTA/pBISA-DL^((ATF-4)) cells weretransfected with mock siRNA or siRNA targeting PKR, PERK, GCN2, or HRIindividually or simultaneously in all combinations (only PKR, PERK, andGCN2 combination is shown). CRL-2813 cells were transfected, in the samemanner except that the transfection mixture also contained the tTAplasmid. Cells were treated with compound #1781 or DMSO and thenormalized F/R ratio was determined by DLR. B)KLN-tTA/pBISA-DL^((ATF-4)) or CRL-2813 cells were transfected withsiRNAs targeting each eIF2α kinases and treated with compound #1781 orDMSO, expression of CHOP mRNA was determined by real-time PCR. C)CRL-2813 cells were transfected with mock or HRI siRNA, treated withcompound #1781 or vehicle, levels of phosphorylated (p-eIF2α) and totaleIF2α (eIF2α) were determined by Western blot. Right panel show thequantification of the western blot. D) KLN-tTA/pBISA-DL^((ATF-4)) cellswere transfected with mock or HRI targeting siRNA, treated with fourN,N′-diarylurea compounds or vehicle and the normalized F/R ratio wasdetermined by DLR. E) CRL-2813 cells were transfected with mock siRNA orHRI siRNA, treated with compound #1781 or vehicle and phosphorylation ofeIF2α was determined by Western blot analysis.

FIGS. 14A and 14B depict that N,N′-diarylurea compounds activate HRI incell free lysates. A and B) Heme supplemented rabbit reticulocyte (a) orin-house prepared human melanoma cancer cell lysates (b) were incubatedwith the indicated concentration of compound 1781 for 30 minutes at 37°C. and phosphorylation of eIF2α was determined by Western blot analysis.Left panels shows quantification of data from three different gels.

FIGS. 15A-15D depict that phosphorylation of eIF2α by HRI mediatesinhibition of cancer cell proliferation by N,N′-diarylureas. A and B)The PC-3 human prostate cancer cells in which endogenous eIF2α isreplaced by recombinant WT or non-phosphorylatable eIF2α-S51A mutantwere treated with the indicated concentrations of N,N′-diarylureas andcell proliferation was measured by SRB assay. Panel a shows the growthinhibition curve for one active (compound #1780) and one inactive(compound #1527) N,N′-diarylurea, panel B shows the calculated IC₅₀ forall four compounds in these genetically engineered cell lines. C)CRL-2813 human melanoma cancer cells were transfected with HRI or mocksiRNA, treated with the indicated concentrations of N,N′-diarylureas andcell proliferation was measured by SRB assay. Panel c shows the growthinhibition curve for one active (compound #1780) and one inactive(compound #1527) N,N′-diarylurea, panel D) shows the calculated IC₅₀ forall four compounds in cells transfected with HRI or mock siRNA.

FIG. 16 depicts that expression of HRI in cancer cell lines correlateswith anti-proliferative activity of N,N′-diarylureas. Lysates wereprepared from mouse KLN squamos cell carcinoma, human CRL-2351 breast,PC-3 prostate, and CRL-2813 melanoma cancer cells, separated by SDS-PAGEand probed with antibodies specific to HRI or β-actin (top panel). TheIC₅₀ of three active N,N′-diarylureas were plotted against the levels ofHRI (corrected for β-actin) in cancer cell lines (lower panel).

FIG. 17 depicts that active N,N′-diarylurea 1781 displays no apparent invivo toxicity. Five female nude mice each were treated with 200 mg/kg,100 mg/kg #1781 in 15 μl DMSO or 15 μl DMSO daily for seven days. Micewere observed daily for signs of toxicity and weighed every other dayfor total of 15 days and then necropsy was performed. The average bodyof each group is plotted against the time.

It will be recognized that the results and examples in the figures areonly illustrative and other examples and illustrations will be readilyrecognized by the person of ordinary skill in the art, given the benefitof this disclosure.

DETAILED DESCRIPTION

In accordance with certain examples, compounds of Formula I and/orFormula II and/or Formula III and/or Formula IV inhibit translation(e.g., translation initiation). Such compounds are useful for thetreatment of (1) proliferative disorders, (2) non-proliferative,degenerative disorders, (3) viral infections, and/or (4) disordersassociated with viral infections. Certain examples are described belowwith reference to various chemical formulae. The chemical formulaereferred to herein can exhibit the phenomena of tautomerism,conformational isomerism, stereo isomerism or geometric isomerism. Asthe formulae drawings within this specification can represent only oneof the possible tautomeric, conformational isomeric, enantiomeric orgeometric isomeric forms, it should be understood that the inventionencompasses any tautomeric, conformational isomeric, enantiomeric orgeometric isomeric forms which exhibit biological or pharmacologicalactivity as described herein.

The compounds and compositions provided below are effective to inhibittranslation (e.g., translation initiation) at least to the extentnecessary for effective treatment of one or more disorders describedherein. While in certain examples translation may be substantiallyinhibited such that little or no activity results, in other examples theinhibition is at least sufficient to relieve and or alleviate thesymptoms from a selected disorder to be treated.

In accordance with certain embodiments, compounds of the invention arerepresented by the generic formula set forth below.

In certain exemplary embodiments with respect to Formula I, II or III,

R₁ is H, Cl, CH₃, OCH₃, NO₂, OH, F, CF₃, OCF₃, Br, CH₃S, AcHN, (CH₃)₂N,CO—NH—NH₂, SO₂NH₂, C(CH₃)₃, COOCH₂CH₃, COCH₃, O(CH₂)₂CH₃, CHO, CO₂H,OCONH₂, CN, C≡CH, N-methylacetamido, 1-[1,2,3]triazolyl,4-[1,2,3]triazolyl, 5-[1,2,3,4]tetrazolyl, guanidine, C₁₋₆-alkyl,C₁₋₆-alkyl amino substituted with: hydroxyl, C₁₋₆-alkoxy, amino, mono-and di-(C₁₋₆-alkyl)amino, carboxy, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylaminocarbonyl, aminosulfonyl, mono- anddi-(C₁₋₆-alkyl)aminosulfonyl, carbamido, mono- anddi-(C₁₋₆-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle,heterocycle, and heteroaryl. C₂₋₆-alkenyl, C₁₋₆-alkoxy, C₂₋₆-alkenyloxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, C₁₋₆-alkylcarbonyloxy,N,N-dimethylamino, N,N-di(C₁₋₆-alkyl)amino, mono- anddi-(C₁₋₆-alkyl)aminocarbonyl, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylsulfonylamino, C₁₋₆-alkylthio, C₁₋₆-alkylsulphinyl, aryl,aryloxy, arylcarbonyl, arylamino, aryl sulfonylamino, hetrocyclyl,hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl,heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, O—(CH₂)₂₋₄-morpholino, O—(CH₂)₂₋₄-(piperazin-1-yl),O—(CH₂)₂₋₄-(4-methylpiperazin-1-yl), O—(CH₂)₂₋₄-mono- anddi-(C₁₋₆-alkyl)amino, O—(CH₂)₂₋₄-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4(1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-(4-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4-(1-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),

R₂ is H, C₁, CH₃, OCH₃, NO₂, OH, F, CF₃, OCF₃, Br, CH₃S, AcHN, (CH₃)₂N,CO—NH—NH₂, SO₂NH₂, C(CH₃)₃, COOCH₂CH₃, COCH₃, O(CH₂)₂CH₃, CHO, CO₂H,OCONH₂, CN, C≡CH, N-methylacetamido, 1-[1,2,3]triazolyl,4-[1,2,3]triazolyl, 5-[1,2, 3, 4]tetrazolyl, guanidine, C₁₋₆-alkyl,C₁₋₆-alkyl amino substituted with: hydroxyl, C₁₋₆-alkoxy, amino, mono-and di-(C₁₋₆-alkyl)amino, carboxy, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylaminocarbonyl, aminosulfonyl, mono- anddi-(C₁₋₆-alkyl)aminosulfonyl, carbamido, mono- anddi-(C₁₋₆-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle,heterocycle, and heteroaryl. C₂₋₆-alkenyl, C₁₋₆-alkoxy, C₂₋₆-alkenyloxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, C₁₋₆-alkylcarbonyloxy,N,N-dimethylamino, N,N-di(C₁₋₆-alkyl)amino, mono- anddi-(C₁₋₆-alkyl)aminocarbonyl, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylsulfonylamino, C₁₋₆-alkylthio, C₁₋₆-alkylsulphinyl, aryl,aryloxy, arylcarbonyl, arylamino, aryl sulfonylamino, hetrocyclyl,hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl,heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, O—(CH₂)₂₋₄-morpholino, O—(CH₂)₂₋₄-(piperazin-1-yl),O—(CH₂)₂₋₄-(4-methylpiperazin-1-yl), O—(CH₂)₂₋₄-mono- anddi-(C₁₋₆-alkyl)amino, O—(CH₂)₂₋₄-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4(1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-(4-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4-(1-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),

R₃ is H, Cl, CH₃, OCH₃, NO₂, OH, F, CF₃, OCF₃, Br, CH₃S, AcHN, (CH₃)₂N,CO—NH—NH₂, SO₂NH₂, C(CH₃)₃, COOCH₂CH₃, COCH₃, O(CH₂)₂CH₃, CHO, CO₂H,OCONH₂, CN, C≡CH, N-methylacetamido, 1-[1,2,3]triazolyl,4-[1,2,3]triazolyl, 5-[1,2,3,4]tetrazolyl, guanidine, C₁₋₆-alkyl,C₁₋₆-alkyl amino substituted with: hydroxyl, C₁₋₆-alkoxy, amino, mono-and di-(C₁₋₆-alkyl)amino, carboxy, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylaminocarbonyl, aminosulfonyl, mono- anddi-(C₁₋₆-alkyl)aminosulfonyl, carbamido, mono- anddi-(C₁₋₆-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle,heterocycle, and heteroaryl. C₂₋₆-alkenyl, C₁₋₆-alkoxy, C₂₋₆-alkenyloxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, C₁₋₆-alkylcarbonyloxy,N,N-dimethylamino, N,N-di(C₁₋₆-alkyl)amino, mono- anddi-(C₁₋₆-alkyl)aminocarbonyl, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylsulfonylamino, C₁₋₆-alkylthio, C₁₋₆-alkylsulphinyl, aryl,aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl,hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl,heteroaryloxy, heteroarylamino, heteroarylcarbonyl,heteroarylsulfonylamino, O—(CH₂)₂₋₄-morpholino,O—(CH₂)₂₋₄-(piperazin-1-yl), O—(CH₂)₂₋₄-(4-methylpiperazin-1-yl),O—(CH₂)₂₋₄-mono- and di-(C₁₋₆-alkyl)amino,O—(CH₂)₂₋₄-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4(1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-(4-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4-(1-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),

R₄ is H, Cl, CH₃, OCH₃, NO₂, OH, F, CF₃, OCF₃, Br, CH₃S, AcHN, (CH₃)₂N,CO—NH—NH₂, SO₂NH₂, C(CH₃)₃, COOCH₂CH₃, COCH₃, O(CH₂)₂CH₃, CHO, CO₂H,OCONH₂, CN, C≡CH, N-methylacetamido, 1-[1,2,3]triazolyl,4-[1,2,3]triazolyl, 5-[1,2,3,4]tetrazolyl, guanidine, C₁₋₆-alkyl,C₁₋₆-alkyl amino substituted with: hydroxyl, C₁₋₆-alkoxy, amino, mono-and di-(C₁₋₆-alkyl)amino, carboxy, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylaminocarbonyl, aminosulfonyl, mono- anddi-(C₁₋₆-alkyl)aminosulfonyl, carbamido, mono- anddi-(C₁₋₆-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle,heterocycle, and heteroaryl. C₂₋₆-alkenyl, C₁₋₆-alkoxy, C₂₋₆-alkenyloxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, C₁₋₆-alkylcarbonyloxy,N,N-dimethylamino, N,N-di(C₁₋₆-alkyl)amino, mono- anddi-(C₁₋₆-alkyl)aminocarbonyl, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylsulfonylamino, C₁₋₆-alkylthio, C₁₋₆-alkylsulphinyl, aryl,aryloxy, arylcarbonyl, arylamino, aryl sulfonylamino, hetrocyclyl,hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl,heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, O—(CH₂)₂₋₄-morpholino, O—(CH₂)₂₋₄-(piperazin-1-yl),O—(CH₂)₂₋₄-(4-methylpiperazin-1-yl), O—(CH₂)₂₋₄-mono- anddi-(C₁₋₆-alkyl)amino, O—(CH₂)₂₋₄-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4(1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-(4-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4-(1-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),

R₅ is H, Cl, CH₃, OCH₃, NO₂, OH, F, CF₃, OCF₃, Br, CH₃S, AcHN, (CH₃)₂N,CO—NH—NH₂, SO₂NH₂, C(CH₃)₃, COOCH₂CH₃, COCH₃, O(CH₂)₂CH₃, CHO, CO₂H,OCONH₂, CN, C≡CH, N-methylacetamido, 1-[1,2,3]triazolyl,4-[1,2,3]triazolyl, 5-[1,2,3,4]tetrazolyl, guanidine, C₁₋₆-alkyl,C₁₋₆-alkyl amino substituted with: hydroxyl, C₁₋₆-alkoxy, amino, mono-and di-(C₁₋₆-alkyl)amino, carboxy, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylaminocarbonyl, aminosulfonyl, mono- anddi-(C₁₋₆-alkyl)aminosulfonyl, carbamido, mono- anddi-(C₁₋₆-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle,heterocycle, and heteroaryl. C₂₋₆-alkenyl, C₁₋₆-alkoxy, C₂₋₆-alkenyloxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, C₁₋₆-alkylcarbonyloxy,N,N-dimethylamino, N,N-di(C₁₋₆-alkyl)amino, mono- anddi-(C₁₋₆-alkyl)aminocarbonyl, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylsulfonylamino, C₁₋₆-alkylthio, C₁₋₆-alkylsulphinyl, aryl,aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl,hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl,heteroaryloxy, heteroarylamino, heteroarylcarbonyl,heteroarylsulfonylamino, O—(CH₂)₂₋₄-morpholino,O—(CH₂)₂₋₄-(piperazin-1-yl), O—(CH₂)₂₋₄-(4-methylpiperazin-1-yl),O—(CH₂)₂₋₄-mono- and di-(C₁₋₆-alkyl)amino,O—(CH₂)₂₋₄-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4(1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-(4-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4-(1-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),

R₆ is H, Cl, CH₃, OCH₃, NO₂, OH, F, CF₃, OCF₃, Br, CH₃S, AcHN, (CH₃)₂N,CO—NH—NH₂, SO₂NH₂, C(CH₃)₃, COOCH₂CH₃, COCH₃, O(CH₂)₂CH₃, CHO, CO₂H,OCONH₂, CN, C≡CH, N-methylacetamido, 1-[1,2,3]triazolyl,4-[1,2,3]triazolyl, 5-[1,2,3,4]tetrazolyl, guanidine, C₁₋₆-alkyl,C₁₋₆-alkyl amino substituted with: hydroxyl, C₁₋₆-alkoxy, amino, mono-and di-(C₁₋₆-alkyl)amino, carboxy, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylaminocarbonyl, aminosulfonyl, mono- anddi-(C₁₋₆-alkyl)aminosulfonyl, carbamido, mono- anddi-(C₁₋₆-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle,heterocycle, and heteroaryl. C₂₋₆-alkenyl, C₁₋₆-alkoxy, C₂₋₆-alkenyloxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, C₁₋₆-alkylcarbonyloxy,N,N-dimethylamino, N,N-di(C₁₋₆-alkyl)amino, mono- anddi-(C₁₋₆-alkyl)aminocarbonyl, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylsulfonylamino, C₁₋₆-alkylthio, C₁₋₆-alkylsulphinyl, aryl,aryloxy, arylcarbonyl, arylamino, aryl sulfonylamino, hetrocyclyl,hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl,heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, O—(CH₂)₂₋₄-morpholino, O—(CH₂)₂₋₄-(piperazin-1-yl),O—(CH₂)₂₋₄-(4-methylpiperazin-1-yl), O—(CH₂)₂₋₄-mono- anddi-(C₁₋₆-alkyl)amino, O—(CH₂)₂₋₄-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4(1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-(4-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4-(1-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),

R₇ is H, Cl, CH₃, OCH₃, NO₂, OH, F, CF₃, OCF₃, Br, CH₃S, AcHN, (CH₃)₂N,CO—NH—NH₂, SO₂NH₂, C(CH₃)₃, COOCH₂CH₃, COCH₃, O(CH₂)₂CH₃, CHO, CO₂H,OCONH₂, CN, C≡CH, N-methylacetamido, 1-[1,2,3]triazolyl,4-[1,2,3]triazolyl, 5-[1,2,3,4]tetrazolyl, guanidine, C₁₋₆-alkyl,C₁₋₆-alkyl amino substituted with: hydroxyl, C₁₋₆-alkoxy, amino, mono-and di-(C₁₋₆-alkyl)amino, carboxy, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylaminocarbonyl, aminosulfonyl, mono- anddi-(C₁₋₆-alkyl)aminosulfonyl, carbamido, mono- anddi-(C₁₋₆-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle,heterocycle, and heteroaryl. C₂₋₆-alkenyl, C₁₋₆-alkoxy, C₂₋₆-alkenyloxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, C₁₋₆-alkylcarbonyloxy,N,N-dimethylamino, N,N-di(C₁₋₆-alkyl)amino, mono- anddi-(C₁₋₆-alkyl)aminocarbonyl, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylsulfonylamino, C₁₋₆-alkylthio, C₁₋₆-alkylsulphinyl, aryl,aryloxy, arylcarbonyl, arylamino, aryl sulfonylamino, hetrocyclyl,hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl,heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, O—(CH₂)₂₋₄-morpholino, O—(CH₂)₂₋₄-(piperazin-1-yl),O—(CH₂)₂₋₄-(4-methylpiperazin-1-yl), O—(CH₂)₂₋₄-mono- anddi-(C₁₋₆-alkyl)amino, O—(CH₂)₂₋₄-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4(1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-(4-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4-(1-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),

R₈ is H, Cl, CH₃, OCH₃, NO₂, OH, F, CF₃, OCF₃, Br, CH₃S, AcHN, (CH₃)₂N,CO—NH—NH₂, SO₂NH₂, C(CH₃)₃, COOCH₂CH₃, COCH₃, O(CH₂)₂CH₃, CHO, CO₂H,OCONH₂, CN, C≡CH, N-methylacetamido, 1-[1,2,3]triazolyl,4-[1,2,3]triazolyl, 5-[1,2,3,4]tetrazolyl, guanidine, C₁₋₆-alkyl,C₁₋₆-alkyl amino substituted with: hydroxyl, C₁₋₆-alkoxy, amino, mono-and di-(C₁₋₆-alkyl)amino, carboxy, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylaminocarbonyl, aminosulfonyl, mono- anddi-(C₁₋₆-alkyl)aminosulfonyl, carbamido, mono- anddi-(C₁₋₆-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle,heterocycle, and heteroaryl. C₂₋₆-alkenyl, C₁₋₆-alkoxy, C₂₋₆-alkenyloxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, C₁₋₆-alkylcarbonyloxy,N,N-dimethylamino, N,N-di(C₁₋₆-alkyl)amino, mono- anddi-(C₁₋₆-alkyl)aminocarbonyl, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylsulfonylamino, C₁₋₆-alkylthio, C₁₋₆-alkylsulphinyl, aryl,aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl,hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl,heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, O—(CH₂)₂₋₄-morpholino, O—(CH₂)₂₋₄-(piperazin-1-yl),O—(CH₂)₂₋₄-(4-methylpiperazin-1-yl), O—(CH₂)₂₋₄-mono- anddi-(C₁₋₆-alkyl)amino, O—(CH₂)₂₋₄-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4(1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-(4-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4-(1-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),

R₉ is H, C₁, CH₃, OCH₃, NO₂, OH, F, CF₃, OCF₃, Br, CH₃S, AcHN, (CH₃)₂N,CO—NH—NH₂, SO₂NH₂, C(CH₃)₃, COOCH₂CH₃, COCH₃, O(CH₂)₂CH₃, CHO, CO₂H,OCONH₂, CN, C≡CH, N-methylacetamido, 1-[1,2,3]triazolyl,4-[1,2,3]triazolyl, 5-[1,2,3,4]tetrazolyl, guanidine, C₁₋₆-alkyl,C₁₋₆-alkyl amino substituted with: hydroxyl, C₁₋₆-alkoxy, amino, mono-and di-(C₁₋₆-alkyl)amino, carboxy, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylaminocarbonyl, aminosulfonyl, mono- anddi-(C₁₋₆-alkyl)aminosulfonyl, carbamido, mono- anddi-(C₁₋₆-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle,heterocycle, and heteroaryl. C₂₋₆-alkenyl, C₁₋₆-alkoxy, C₂₋₆-alkenyloxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, C₁₋₆-alkylcarbonyloxy,N,N-dimethylamino, N,N-di(C₁₋₆-alkyl)amino, mono- anddi-(C₁₋₆-alkyl)aminocarbonyl, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylsulfonylamino, C₁₋₆-alkylthio, C₁₋₆-alkylsulphinyl, aryl,aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl,hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl,heteroaryloxy, heteroarylamino, heteroarylcarbonyl,heteroarylsulfonylamino, O—(CH₂)₂₋₄-morpholino,O—(CH₂)₂₋₄-(piperazin-1-yl), O—(CH₂)₂₋₄-(4-methylpiperazin-1-yl),O—(CH₂)₂₋₄-mono- and di-(C₁₋₆-alkyl)amino,O—(CH₂)₂₋₄-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4(1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-(4-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4-(1-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),

R₁₀ is H, Cl, CH₃, OCH₃, NO₂, OH, F, CF₃, OCF₃, Br, CH₃S, AcHN, (CH₃)₂N,CO—NH—NH₂, SO₂NH₂, C(CH₃)₃, COOCH₂CH₃, COCH₃, O(CH₂)₂CH₃, CHO, CO₂H,OCONH₂, CN, C≡CH, N-methylacetamido, 1-[1,2,3]triazolyl,4-[1,2,3]triazolyl, 5-[1,2,3,4]tetrazolyl, guanidine, C₁₋₆-alkyl,C₁₋₆-alkyl amino substituted with: hydroxyl, C₁₋₆-alkoxy, amino, mono-and di-(C₁₋₆-alkyl)amino, carboxy, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylaminocarbonyl, aminosulfonyl, mono- anddi-(C₁₋₆-alkyl)aminosulfonyl, carbamido, mono- anddi-(C₁₋₆-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle,heterocycle, and heteroaryl. C₂₋₆-alkenyl, C₁₋₆-alkoxy, C₂₋₆-alkenyloxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, C₁₋₆-alkylcarbonyloxy,N,N-dimethylamino, N,N-di(C₁₋₆-alkyl)amino, mono- anddi-(C₁₋₆-alkyl)aminocarbonyl, C₁₋₆-alkylcarbonylamino,C₁₋₆-alkylsulfonylamino, C₁₋₆-alkylthio, C₁₋₆-alkylsulphinyl, aryl,aryloxy, arylcarbonyl, arylamino, aryl sulfonylamino, hetrocyclyl,hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl,heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, O—(CH₂)₂₋₄-morpholino, O—(CH₂)₂₋₄-(piperazin-1-yl),O—(CH₂)₂₋₄-(4-methylpiperazin-1-yl), O—(CH₂)₂₋₄-mono- anddi-(C₁₋₆-alkyl)amino, O—(CH₂)₂₋₄-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4(1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-(4-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),O—(CH₂)₂₋₄-4-(1-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl),

R₁₁ is H, CH₃

R₁₂ is H, CH₃,

R₁₃ is O, S, NH or NR₁₉. For compounds of Formulae II and III, R₁₃ ispreferably S, NH or NR₁₉.

R₁₄ is NH, S or

R₁₅ is NH, S or NHNCH.

R₁₆ is C.

R₁₇ is H, CH₃, —[(CH₂)₂—O]₁₋₃H, —[(CH₂)₂—O]₁₋₃CH₃, —(CH₂)₂—NH₂,—(CH₂)—NHCH₃, —(CH₂)₂—N(CH₃)₂, —[(CH₂)₂—O]₁₋₂(CH₂)₂—NHCH₃,—[(CH₂)₂—O]₁₋₂(CH₂)₂—N(CH₃)₂,

R₁₈ is H, CH₃, —[(CH₂)₂—O]₁₋₃H, —[(CH₂)₂—O]₁₋₃CH₃, —(CH₂)₂—NH₂,—(CH₂)₂—NHCH₃, —(CH₂)₂—N(CH₃)₂, —[(CH₂)₂—O]₁₋₂(CH₂)₂—NHCH₃,—[(CH₂)₂—O]₁₋₂(CH₂)₂—N(CH₃)₂,

R₁₉ is CH₃, C(CH₃)₃, C₁₋₆-alkyl, C₁₋₆-alkyl substituted with: hydroxyl,C₁₋₆-alkoxy, amino, mono- and di-(C₁₋₆-alkyl)amino, carboxy,C₁₋₆-alkylcarbonylamino, C₁₋₆-alkylaminocarbonyl, aminosulfonyl, mono-and di-(C₁₋₆-alkyl)aminosulfonyl, carbamido, mono- anddi-(C₁₋₆-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle,heterocycle, and heteroaryl. C₂₋₆-alkenyl, —(CH₂)₂₋₄-morpholino,—(CH₂)₂₋₄-(piperazin-1-yl), —(CH₂)₂₋₄-(4-methylpiperazin-1-yl),—(CH₂)₂₋₄-mono- and di-(C₁₋₆-alkyl)amino,—(CH₂)₂₋₄-1H-[1,2,3]triazol-1-yl, —(CH₂)₂₋₄-1H-[1,2,3]triazol-4-yl,—(CH₂)₂₋₄-(4-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-1-yl), or—(CH₂)₂₋₄-(1-(C₁₋₆-alkyl)-1H-[1,2,3]triazol-4-yl).

According to one particular embodiment of Formula I, II or III, R₁₁ andR₁₂ are absent and a covalent linkage is present between the nitrogenesthat is

According to another particular embodiment R₁₇ and R₁₈ are absent andreplaced by either

covalently linked to the nitrogen atom.

In certain exemplary embodiments, compounds within the scope of FormulaI, II or III are those where, optionally, at least one atom iscovalently linked between two R groups. In certain exemplaryembodiments, a covalent linkage is present between R₁ and R₁₅ that is

In certain exemplary embodiments, a covalent linkage is present betweenR₂ and R₃ that is

In other embodiments, a covalent linkage is present between R₇ and R₈that is

In certain exemplary embodiments, a covalent linkage is present betweenR₁ and R₂ that is

In certain exemplary embodiments, a covalent linkage is present betweenR₆ and R₇ that is

In other embodiments, a covalent linkage is present between R₈ and R₉that is

In other embodiments, a covalent linkage is present between R₉ and R₁₀that is

In other embodiments, a covalent linkage is present between R₁₀ and R₁₂that is

In other embodiments, a covalent linkage is present between R₁₄ and R₁₅that is

In other embodiments, a covalent linkage is present between R₁₅ and R₁₆that is

With respect to the substituents identified above, it is to beunderstood that the substituents are to be covalently linked to an atomor atoms and so one of skill in the art would understand that theterminal lines in the moieties for the various R groups in thisapplication may indicate linkage points to an atom and not the presenceof an atom itself.

In accordance with certain embodiments, compounds of the invention arerepresented by the generic formula set forth below.

In certain exemplary embodiments with respect to Formula IV above,

R₁ is S or O.

R₂ is NH₂, CH₃,

and

R₃ is OH, NH₂, CH₃,

Specific compounds within the scope of the present invention include thefollowing/those set forth in Table 1, below.

TABLE 1 Compounds according to certain exemplary embodiments. CompoundNumber Structure 1439

1440

1441

1442

1443

1444

1445

1446

1447

1448

1449

1450

1451

1452

1453

1454

1474

1475

1476

1477

1478

1479

1480

1481

1482

1483

1484

1485

1486

1487

1496

1497

1498

1499

1500

1501

1502

1503

1504

1505

1506

1507

1508

1509

1510

1511

1518

1519

1520

1521

1522

1523

1524

1525

1526

1527

1528

1529

1530

1531

1532

1533

1534

1535

1536

1537

1538

1539

1540

1541

1542

1543

1544

1545

1546

1547

1548

1549

1550

1551

1552

1553

1554

1555

1556

1557

1558

1559

1560

1561

1562

1563

1564

1565

1566

1567

1568

1569

1570

1571

1572

1573

1574

1575

1576

1577

1578

1584

1585

1586

1587

1612

1613

BAS 93909

BAS 93826

BAS 167472

BAS 728878

BTB 06969

CD 06116

HTS 02561

HTS 02562

HTS 04043

HTS 06049

KM 08479

KM 09745

KM 09748

KM 09749

KM 09750

RF 00386

RF 00680

S 09172

SPB 06399

BAS 4320322

CGX-0138398

CGX-0778260

CGX-0778468

CGX-0778832

CGX-0778728

CGX-0778220

CGX-0778272

CGX-0778480

CGX-0778688

CGX-0778636

CGX-0778844

CGX-0779582

CGX-0790347

CGX-0790503

CGX-0778896

CGX-2086541

CGX-3075570

BAY 43-9006

GK 00687

GK 00700

T1653

T1649

T1650

T1651

T1652

T1654

T1655

T1656

T1657

1658

1659

1660

1661

1662

1663

1664

1665

1778

1779

1780

1781

1782

1783

1791

1792

1793

1794

1797

1798

1799

1800

1801

1802

1803

1804

1805

1806

1809

1810

1811

1812

1813

1822

1823

BLS17

BLS13

In at least certain examples, the compounds disclosed here can be usedin the treatment of cellular proliferative disorders, such as cancer andnon-cancerous cellular proliferative disorders. Treatment of cellularproliferative disorders is intended to include, but is not limited to,inhibition of proliferation including rapid proliferation. As usedherein, the term “cellular proliferative disorder” includes, but is notlimited to, disorders characterized by undesirable or inappropriateproliferation of one or more subset(s) of cells in a multicellularorganism. The term “cancer” refers to various types of malignantneoplasms, most of which can invade surrounding tissues, and maymetastasize to different sites (see, for example, PDR Medical Dictionary1st edition (1995)). The terms “neoplasm” and “tumor” refer to anabnormal tissue that grows by cellular proliferation more rapidly thannormal and continues to grow after the stimuli that initiatedproliferation is removed. Id. Such abnormal tissue shows partial orcomplete lack of structural organization and functional coordinationwith the normal tissue which may be either benign (i.e., benign tumor)or malignant (i.e., malignant tumor).

Examples of general categories of cancer include, but are not limitedto, carcinomas (i.e., malignant tumors derived from epithelial cellssuch as, for example, common forms of breast, prostate, lung and coloncancer), sarcomas (i.e., malignant tumors derived from connective tissueor mesenchymal cells), lymphomas (i.e., malignancies derived fromhematopoietic cells), leukemias (i.e., malignancies derived fromhematopoietic cells), germ cell tumors (i.e., tumors derived fromtotipotent cells. In adults most often found in the testicle or ovary;in fetuses, babies and young children, most often found on the bodymidline, particularly at the tip of the tailbone), blastic tumors (i.e.,a typically malignant tumor which resembles an immature or embryonictissue) and the like. One of skill in the art will understand that thislist is exemplary only and is not exhaustive, as one of skill in the artwill readily be able to identify additional cancers based on thedisclosure herein.

Examples of specific neoplasms intended to be encompassed by the presentinvention include, but are not limited to, acute lymphoblastic leukemia;myeloid leukemia, acute myeloid leukemia, childhood; adrenocorticalcarcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer;appendix cancer; astrocytoma (e.g., cerebellar, cerebral); atypicalteratoid/rhabdoid tumor; basal cell carcinoma; bile duct cancer,extrahepatic; bladder cancer; bone cancer, osteosarcoma and malignantfibrous histiocytoma; brain tumor (e.g., brain stem glioma, centralnervous system atypical teratoid/rhabdoid tumors, central nervous systemembryonal tumors, cerebellar astrocytoma, cerebral astrocytoma/malignantglioma, craniopharyngioma, ependymoblastoma, ependymoma,medulloblastoma, medulloepithelioma, pineal parenchymal tumors ofintermediate differentiation, supratentorial primitive neuroectodermaltumors and/or pineoblastoma, visual pathway and/or hypothalamic glioma,brain and spinal cord tumors); breast cancer; bronchial tumors; Burkittlymphoma; carcinoid tumor (e.g., gastrointestinal); carcinoma of unknownprimary; central nervous system (e.g., atypical teratoid/rhabdoid tumor,embryonal tumors (e.g., lymphoma, primary); cerebellar astrocytoma;cerebral astrocytoma/malignant glioma; cervical cancer; chordoma;chronic lymphocytic leukemia; chronic myelogenous leukemia; chronicmyeloproliferative disorders; colon cancer; colorectal cancer;craniopharyngioma; cutaneous T-cell lymphoma; embryonal tumors, centralnervous system; endometrial cancer; ependymoblastoma; ependymoma;esophageal cancer; Ewing family of tumors; extracranial germ cell tumor;extragonadal germ cell tumor; extrahepatic bile duct cancer; eye cancer(e.g., intraocular melanoma, retinoblastoma); gallbladder cancer;gastric cancer; gastrointestinal tumor (e.g., carcinoid tumor, stromaltumor (gist), stromal cell tumor); germ cell tumor (e.g., extracranial,extragonadal, ovarian); gestational trophoblastic tumor; glioma (e.g.,brain stem, cerebral astrocytoma); hairy cell leukemia; head and neckcancer; hepatocellular cancer; Hodgkin lymphoma; hypopharyngeal cancer;hypothalamic and visual pathway glioma; intraocular melanoma; islet celltumors; Kaposi sarcoma; kidney cancer; large cell tumors; laryngealcancer (e.g., acute lymphoblastic, acute myeloid); leukemia (e.g., acutemyeloid, chronic lymphocytic, chronic myelogenous, hairy cell); lipand/or oral cavity cancer; liver cancer; lung cancer (e.g., non-smallcell, small cell); lymphoma (e.g., AIDS-related, Burkitt, cutaneousTcell, Hodgkin, non-Hodgkin, primary central nervous system);macroglobulinemia, Waldenstrom; malignant fibrous histiocytoma of boneand/or osteosarcoma; medulloblastoma; medulloepithelioma; melanoma;merkel cell carcinoma; mesothelioma; metastatic squamous neck cancer;mouth cancer; multiple endocrine neoplasia syndrome; multiplemyeloma/plasma cell neoplasm; mycosis fungoides; myelodysplasticsyndromes; myelodysplastic/myeloproliferative diseases; myelogenousleukemia (e.g., chronic, acute, multiple); myeloproliferative disorders,chronic; nasal cavity and/or paranasal sinus cancer; nasopharyngealcancer; neuroblastoma; non-Hodgkin lymphoma; non-small cell lung cancer;oral cancer; oral cavity cancer, oropharyngeal cancer; osteosarcomaand/or malignant fibrous histiocytoma of bone; ovarian cancer (e.g.,ovarian epithelial cancer, ovarian germ cell tumor, ovarian lowmalignant potential tumor); pancreatic cancer (e.g., islet cell tumors);papillomatosis; paranasal sinus and/or nasal cavity cancer; parathyroidcancer; penile cancer; pharyngeal cancer; pheochromocytoma; pinealparenchymal tumors of intermediate differentiation; pineoblastoma andsupratentorial primitive neuroectodermal tumors; pituitary tumor; plasmacell neoplasm/multiple myeloma; pleuropulmonary blastoma; primarycentral nervous system lymphoma; prostate cancer; rectal cancer; renalcell cancer; renal, pelvis and/or ureter, transitional cell cancer;respiratory tract carcinoma involving the nut gene on chromosome 15;retinoblastoma; rhabdomyosarcoma; salivary gland cancer; sarcoma (e.g.,Ewing family of tumors, Kaposi, soft tissue, uterine); Sezary syndrome;skin cancer (e.g., non-melanoma, melanoma, merkel cell); small cell lungcancer; small intestine cancer; soft tissue sarcoma; squamous cellcarcinoma; squamous neck cancer with occult primary, metastatic; stomachcancer; supratentorial primitive neuroectodermal tumors; T-celllymphoma, cutaneous; testicular cancer; throat cancer; thymoma and/orthymic carcinoma; thyroid cancer; transitional cell cancer of the renal,pelvis and/or ureter; trophoblastic tumor; unknown primary sitecarcinoma; urethral cancer; uterine cancer, endometrial; uterinesarcoma; vaginal cancer; visual pathway and/or hypothalamic glioma;vulvar cancer; Waldenstrom macroglobulinemia; Wilms tumor and the like.For a review, see the National Cancer Institute's Worldwide Website(cancer.gov/cancertopics/alphalist). One of skill in the art willunderstand that this list is exemplary only and is not exhaustive, asone of skill in the art will readily be able to identify additionalcancers and/or neoplasms based on the disclosure herein.

Examples of noncancerous cellular proliferative disorders includesfibroadenoma, adenoma, intraductal papilloma, nipple adenoma, adenosis,fibrocystic disease or changes of breast, plasma cell proliferativedisorder (PCPD), restenosis, atherosclerosis, rheumatoid arthritis,myofibromatosis, fibrous hamartoma, granular lymphocyte proliferativedisorders, benign hyperplasia of prostate, heavy chain diseases (HCDs),lymphoproliferative disorders, psoriasis, idiopathic pulmonary fibrosis,sclroderma, cirrhosis of the liver, IgA nephropathy, mesangialproliferative glomerulonephritis, membranoproliferativeglomerulonephritis, hemangiomas, vascular and non-vascular intraocularproliferative disorders and the like. One of skill in the art willunderstand that this list is exemplary only and is not exhaustive, asone of skill in the art will readily be able to identify additionalnoncancerous cellular proliferative disorders based on the disclosureherein.

The language “treatment of cellular proliferative disorders” is intendedto include, but is not limited to, the prevention of the growth ofneoplasms in a subject or a reduction in the growth of pre-existingneoplasms in a subject, as well as the prevention or reduction ofincreased or uncontrollable cell growth. The inhibition also can be theinhibition of the metastasis of a neoplasm from one site to another. Incertain embodiments, the neoplasms are sensitive to one or morecompounds of Formulae I and II as described herein.

In accordance with certain other examples, methods for treating viralinfections are also disclosed. Treatment of viral infections is intendedto include, but is not limited to, the use of a N,N′-diarylurea and/orN,N′-diarylthiourea compounds described herein to prevent the initiationof viral protein synthesis. The term “viral infection,” as used herein,refers to one or more cells which have been infected with a virus, suchas a DNA or RNA animal virus. As used herein, RNA viruses include, butare not limited to, virus families such as picornaviridae (e.g.,polioviruses), reoviridae (e.g., rotaviruses), togaviridae (e.g.,encephalitis viruses, yellow fever virus, rubella virus),orthomyxoviridae (e.g., influenza viruses), paramyxoviridae (e.g.,respiratory syncytial virus, measles virus, mumps virus, parainfluenzavirus), rhabdoviridae (e.g., rabies virus), coronaviridae, bunyaviridae,flaviviridae, filoviridae, arenaviridae, bunyaviridae, and retroviridae(e.g., human T-cell lymphotropic viruses (HTLV), human immunodeficiencyviruses (HIV)). As used herein, DNA viruses include, but are not limitedto, virus families such as papovaviridae (e.g., papilloma viruses),adenoviridae (e.g., adenovirus), herpesviridae (e.g., herpes simplexviruses), and poxviridae (e.g., variola viruses). In certainembodiments, the viral infection is caused by hepatitis B virus,hepatitis C virus and/or HIV. One of skill in the art will understandthat this list is exemplary only and is not exhaustive, as one of skillin the art will readily be able to identify additional viral infectionsbased on the disclosure herein.

In accordance with other examples, methods for treating disordersassociated with viral infections are disclosed. Treatment of one or moredisorders associated with viral infections is intended to include, butis not limited to, the use of a N,N′-diarylurea and/orN,N′-diarylthiourea compound described herein to reduce or alleviate oneor more symptoms of a viral infection. As used herein, the term“disorders associated with viral infection” refers to the host'sresponse to infection by one or more viruses. Such responses include,but are not limited to neurological symptoms (e.g., encephalitis,meningoencephalitis, paralysis, myelopathy, neuropathy, asepticmeningitis, hemiparesis, dementia, dysphagia, lack of muscularcoordination, impaired vision, coma, and the like), wasting symptoms(e.g., inflammatory cell infiltration, perivascular cuffing of bloodvessels, demyelination, necrosis, reactive gliosis and the like),gastroenteritis symptoms (e.g., diarrhea, vomiting, cramps and thelike), hepatitis symptoms (nausea, vomiting, right upper quadrant pain,raised liver enzyme levels (e.g., AST, ALT and the like), jaundice andthe like), hemorrhagic fever symptoms (e.g., headache, fever, chillsbody pains, diarrhea, vomiting, dizziness, confusion, abnormal behavior,pharyngitis, conjunctivitis, red face, red neck, hemorrhage, organfailure and the like), oncogenic symptoms (e.g., sarcomas, leukemias andthe like, as well as “rare” malignancies, e.g., Kaposi's sarcoma, oralhairy leukoplasia, lymphomas and the like), immunodeficiency symptoms(e.g., opportunistic infections, wasting, rare malignancies,neurological disease, fever, diarrhea, skin rashes and the like),lesions (e.g., warts (e.g., common wart, flat wart, deep hyperkaratoticpalmoplantar wart, superficial mosaic type palmoplantar wart and thelike), epidermodysplasia, mucosal lesions, ulcers and the like), andsystemic symptoms (e.g., fever, chills, headache, muscle pain, bonepain, joint pain, pharyngitis, tonsillitis, sinusitis, otitis,bronchitis, pneumonia, bronchopneumonia, nausea, vomiting, increasedsalivation, rash, macules, lymphadenopothy, arthritis, ulcers,photosensitivity, weight loss, irritability, restlessness, anxiety,coma, death and the like). Disorders associated with viral infectionsare described in Fields Virology 4^(th) Ed. (2001) Lippincott, Williams& Wilkins, and the introduction to medical virology website(web.uct.ac.za/depts./mmi/jmoodie/introvi2.html). One of skill in theart will understand that this list is exemplary only and is notexhaustive, as one of skill in the art will readily be able to identifyadditional disorders associate with viral infections based on thedisclosure herein.

In accordance with other examples, methods for treating disorderscharacterized by unwanted synthesis and/or abnormal accumulation of oneor more mutant and/or wild-type proteins are provided. Treatment of oneor more disorders associated with unwanted synthesis and/or abnormalaccumulation is intended to include, but is not limited to, the use of aN,N′-diarylurea and/or N,N′-diarylthiourea compound described herein toreduce or alleviate one or more symptoms characterized by unwantedsynthesis and/or abnormal accumulation. Without intending to be bound byscientific theory, contacting a subject afflicted with a disordercharacterized by unwanted synthesis and/or abnormal accumulation of oneor more mutant and/or wild-type proteins with a compound describedherein (e.g., a compound that can inhibit translation initiation) canreduce the load on the protein-folding machinery and, accordingly, mayreduce the severity of the disorder. Disorders associated with unwantedsynthesis and/or abnormal accumulation of one or more mutant and/orwild-type proteins include, but are not limited to, Tay-Sachs disease,cystic fibrosis, phenylketonuria, Fabry disease, Alzheimer's disease,Huntington's disease, Parkinson's disease, congophilic angiopathy, prionrelated disorders (i.e., transmissible spongiform encephalopathies suchas Creutzfeldt-Jacob disease, kuru, fatal familial insomnia, scrapie,bovine spongiform encephalopathy and the like) and the like. One ofskill in the art will understand that this list is exemplary only and isnot exhaustive, as one of skill in the art will readily be able toidentify additional disorders characterized by unwanted synthesis and/orabnormal accumulation of one or more mutant and/or wild-type proteinsbased on the disclosure herein.

In accordance with other examples, methods for treatingnon-proliferative, degenerative disorders associated with aberranttranslation initiation using a N,N′-diarylurea and/orN,N′-diarylthiourea compound described herein to alleviate and/or reduceone or more symptoms associated with a non-proliferative, degenerativedisorder are disclosed. Treatment of non-proliferative, degenerativediseases is intended to include, but is not limited to, the use ofN,N′-diarylurea and/or N,N′-diarylthiourea compounds described herein.As used herein, the term “non-proliferative degenerative disorder” isintended to include, but is not limited to, diseases characterized by aloss of function of cells, tissues, and/or organs due to aberranttranslation initiation. Non-proliferative degenerative disordersinclude, but are not limited to, disorders such as Alzheimer's diseaseand insulin resistance. One of skill in the art will understand thatthis list is exemplary only and is not exhaustive, as one of skill inthe art will readily be able to identify additional non-proliferativedegenerative disorders based on the disclosure herein.

In accordance with certain other examples, kits for treating one or more(1) proliferative disorders, (2) non-proliferative, degenerativedisorders, (3) viral infections, and/or (4) disorders associated withviral infections are provided. In one example, the kit may comprise oneor more compounds of Formulae I and II as described herein. In anotherexample, the kit may comprise a pharmaceutically acceptable carrier. Inan additional example, the kit may also include instructions fortreating (1) proliferative disorders, (2) non-proliferative,degenerative disorders, (3) viral infections, (4) disorders associatedwith viral infections, and/or (5) disorders characterized by unwantedprotein synthesis or diseases for which reducing protein synthesis isadvantageous. In some examples, the kit may also comprise, e.g., abuffering agent, a preservative, or a protein stabilizing agent. Inother examples, the kit may also contain a control sample or a series ofcontrol samples which can be assayed and compared to the test samplecontained. Other suitable components for including in the kit will beselected by the person of ordinary skill in the art, given the benefitof this disclosure.

In accordance with certain examples, compounds of the present inventioncan be incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the compoundsdisclosed here and a pharmaceutically acceptable carrier. As used hereinthe term “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

In accordance with certain examples, a pharmaceutical composition of theinvention is formulated to be compatible with its intended route ofadministration. Such pharmaceutical compositions may be administered byinhalation, transdermally, orally, rectally, transmucosally,intestinally, parenterally, intramuscularly, subcutaneously,intravenously or other suitable methods that will be readily selected bythe person of ordinary skill in the art, given the benefit of thisdisclosure. For example, solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerin, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

In accordance with other examples, pharmaceutical compositions suitablefor injectable use include sterile aqueous solutions (where watersoluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. Forintravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, CREMPHOR EL™ (BASF, Parsippany, N.J.), orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

In accordance with other examples, sterile injectable solutions can beprepared by incorporating the active compound in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions,methods of preparation can be vacuum drying and freeze-drying whichyields a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof. Oralcompositions generally include an inert diluent or an edible carrier.They can be enclosed in gelatin capsules or compressed into tablets. Forthe purpose of oral therapeutic administration, the active compound canbe incorporated with excipients and used in the form of tablets,troches, or capsules. Oral compositions can also be prepared using afluid carrier for use as a mouthwash, wherein the compound in the fluidcarrier is applied orally and swished and expectorated or swallowed.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In at least certain examples, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, incorporated herein by reference in its entirety for allpurposes.

In accordance with certain examples, pharmaceutical compositions of theinvention comprise one or more N,N′-diarylurea and/orN,N′-diarylthiourea compounds covalently linked to a peptide (i.e., apolypeptide comprising two or more amino acids). Peptides may beassembled sequentially from individual amino acids or by linkingsuitable small peptide fragments. In sequential assembly, the peptidechain is extended stepwise, starting at the C-terminus, by one aminoacid per step. In fragment coupling, fragments of different lengths canbe linked together, and the fragments can also be obtained by sequentialassembly from amino acids or by fragment coupling of still shorterpeptides.

In both sequential assembly and fragment coupling it is necessary tolink the units (e.g., amino acids, peptides, compounds and the like) byforming an amide linkage, which can be accomplished via a variety ofenzymatic and chemical methods. The methods described herein forformation of peptidic amide linkages are also suitable for the formationof non-peptidic amide linkages.

Chemical methods for forming the amide linkage are described in detailin standard references on peptide chemistry, including Muller, Methodender organischen Chemie Vol. XV/2, 1-364, Thieme Verlag, Stuttgart,(1974); Stewart and Young, Solid Phase Peptide Synthesis, 31-34 and71-82, Pierce Chemical Company, Rockford, Ill. (1984); Bodanszky et al.,Peptide Synthesis, 85-128, John Wiley & Sons, New York, (1976); Practiceof Peptide Synthesis, M. Bodansky, A. Bodansky, Springer-Verlag, 1994and other standard works in peptide chemistry. Methods include the azidemethod, the symmetric and mixed anhydride method, the use of in situgenerated or preformed active esters, the use of urethane protectedN-carboxy anhydrides of amino acids and the formation of the amidelinkage using coupling reagents, such as dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide (DIC),1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), pivaloylchloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDCl), n-propane-phosphonic anhydride (PPA), N,N-bis(2-oxo-3-oxazolidinyl)amido phosphoryl chloride (BOP-Cl),bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBrop),diphenylphosphoryl azide (DPPA), Castro's reagent (BOP, PyBop),O-benzotriazolyl-N,N,N′,N′-tetramethyluronium salts (HBTU),O-azabenzotriazolyl-N,N,N′,N′-tetramethyluronuim salts (TATU),diethylphosphoryl cyanide (DEPCN),2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (Steglich'sreagent; HOTDO), 1,1′-carbonyldiimidazole (CDI) and the like. Thecoupling reagents can be employed alone or in combination with additivessuch as N,N-dimethyl-4-aminopyridine (DMAP), N-hydroxy-benzotriazole(HOBt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu),2-hydroxypyridine and the like.

In accordance with other examples, methods of modulating translationinitiation for therapeutic purposes are disclosed. In one example, amethod involves contacting a cell with an agent that inhibitstranslation initiation. An agent that inhibits translation initiationcan be any one of the compounds described herein, such as aN,N′-diarylurea and/or N,N′-diarylthiourea compound. In at least certainexamples, the compound modulates the depletion of intracellular calciumstores. Methods of modulating translation initiation can be performed invitro (e.g., by culturing a cell with the agent) or, alternatively, invivo (e.g., by administering the agent to a subject). Certain examplesdisclosed herein are directed to methods of treating an individualafflicted with a disease or disorder characterized by aberranttranslation initiation. Examples of such disorders are described herein.In one embodiment, the method involves administering an agent (e.g., anagent identified by a screening assay described herein), or combinationof agents that inhibits translation initiation. As used herein, anindividual afflicted with a disease or disorder is intended to includeboth human and non-human mammals. Examples of non-human mammals include,but are not limited to, non-human primates, horses, cows, goats, sheep,dogs, cats, mice, rats, hamsters, guinea pigs and the like.

The present invention provides for both prophylactic and therapeuticmethods of treating a subject for one or more (1) proliferativedisorders, (2) non-proliferative, degenerative disorders, (3) viralinfections, and/or (4) disorders associated with viral infection. In oneaspect, the invention provides a method for preventing in a subject, adisease or condition associated with one or more (1) proliferativedisorders, (2) non-proliferative, degenerative disorders, (3) viralinfections, and/or (4) disorders associated with viral infection, byadministering, to the subject one or more N,N′-diarylurea and/orN,N′-diarylthiourea compounds described herein to modulate one or more(1) proliferative disorders, (2) non-proliferative, degenerativedisorders, (3) viral infections, and/or (4) disorders associated withviral infection. Administration of a prophylactic agent can occur priorto the manifestation of symptoms, such that a disease or disorder isprevented or, alternatively, delayed in its progression.

Another aspect of the invention pertains to therapeutic methods oftreating one or more (1) proliferative disorders, (2) non-proliferative,degenerative disorders, (3) viral infections, and/or (4) disordersassociated with viral infection for therapeutic purposes. Accordingly,in an exemplary embodiment, a therapeutic method of the inventioninvolves contacting a subject with a N,N′-diarylurea and/orN,N′-diarylthiourea compound that therapeutically treats one or more (1)proliferative disorders, (2) non-proliferative, degenerative disorders,(3) viral infections, (4) disorders associated with viral infection,and/or (5) disorders characterized by unwanted protein synthesis ordiseases for which reducing protein synthesis is advantageous.

One embodiment of the present invention involves a method of treating atranslation initiation-associated disease or disorder which includes thestep of administering a therapeutically and/or prophylacticallyeffective amount of an agent which inhibits translation initiation to asubject. In another embodiment, a subject is administered atherapeutically and/or prophylactically effective amount that iseffective to deplete intracellular calcium stores. As defined herein, atherapeutically and/or prophylactically effective amount of agent (i.e.,an effective dosage) ranges from about 0.001 to 30 mg/kg body weight,from about 0.01 to 25 mg/kg body weight, from about 0.1 to 20 mg/kg bodyweight, from about 1 to 10 mg/kg, from about 2 to 9 mg/kg, from about 3to 8 mg/kg, from about 4 to 7 mg/kg, or from about 5 to 6 mg/kg bodyweight. The skilled artisan will appreciate that certain factors mayinfluence the dosage required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Treatment of a subject with a therapeutically and/orprophylactically effective amount of an inhibitor can include a singletreatment or can include a series of treatments. It will also beappreciated that the effective dosage of in used for treatment mayincrease or decrease over the course of a particular treatment.

It is to be understood that the embodiments of the present inventionwhich have been described are merely illustrative of some of theapplications of the principles of the present invention. Numerousmodifications may be made by those skilled in the art based upon theteachings presented herein without departing from the true spirit andscope of the invention. The contents of all references, patents andpublished patent applications cited throughout this application arehereby incorporated by reference in their entirety for all purposes.

The following examples are set forth as being representative of thepresent invention. These examples are not to be construed as limitingthe scope of the invention as these and other equivalent embodimentswill be apparent in view of the present disclosure, figures, andaccompanying claims.

Example I N,N′-Diarylurea Translation Initiation Inhibitors

Design and Development of a Ternary Complex Assay

For assay development, a bi-directional plasmid was designed in which acommon promoter/enhancer complex drives the transcription of fireflyluciferase (F-luc) ORF fused to the 5′ untranslated region (UTR) ofATF-4, and of the renilla luciferase (R-luc) ORF fused to a90-nucleotide 5′ UTR derived from the plasmid (FIG. 6A). Because thetetracycline-regulated transactivator ((tTA), required for drivingtranscription from this vector) is not normally expressed in themammalian cells, stable KLN cancer cells were constructed that expressedtTA (KLN-tTA).

The KLN-tTA colonies that drove expression of reporter genes frompBISA-DL plasmid were selected by transient transfection and dualluciferase assay. One of these KLN-tTA cell lines was transfected withthe pBISA-DL^((ATF-4)) expression vector, stable colonies were selectedby dual luciferase assay and expanded. To determine if reducedavailability of the eIF2*GTP*Met-tRNA_(i) ternary complex increased thetranslation of firefly luciferase and decreased translation of renillaluciferase, selected KLN-tTA/pBISA-DL^((AFT-4)) colonies were treatedwith thapsigargin (TG) or tunicamycin (TU), two agents known to causephosphorylation of eIF2α. FIG. 6B shows that treatment with these agentsincreased the expression of firefly luciferase and decreased expressionof renilla luciferase, leading to an increase in the ratio of fireflyactivity relative to renilla activity. This effect was due to presenceof multiple uORFs but not other elements in the 5′ UTR of ATF-4 (forexample an internal ribosomal entry site, IRES, element) because removalof uORF2 by insertion of a single nucleotide abolished increasedfirefly/renilla ratio in TG or TU treated cells (FIG. 6C). Furthermore,the firefly/renilla ratio was increased only in response to decreasedabundance of the ternary complex but not inhibition of cell growthbecause several anti-proliferative agents such as etoposite or mitomycinhad no effect on Firefly luciferase/Renilla luciferase ratio, indicatingthat this assay is suitable for identification of agents that reduceabundance of the ternary complex (Table 6).

This assay was then adapted for high throughput screening in 96 and 384well plates. This was done by evaluating the cell density, length ofexposure to compounds, DMSO tolerance and optimum firefly and renillasubstrate. We then challenged these cells with thapsigargin or DMSO. Thescattered plot of these data is shown in FIG. 6D. Using these data, thesuitability of the assay for high throughput screening in 384 well wasdetermined by determining signal to background ratio and the Z-factor.Cell-based assays usually have higher variation than homogeneous invitro assays due to position effect in the plate, and other variablesassociated with handling of cells and the plates. Overall, this assayhad a very high signal to background ratio (approximately 100 for bothluciferases), and a Z score of 0.58, an excellent value for a cell basedassay.

Screening

Screening was conducted in 384 well white opaque plates (Nalge Nunc),100 μl volume RPMI+10% fetal bovine serum. Cells were plated at thesub-confluent density of 10,000 cells/well, and allowed to attach for aperiod of 16-18 hours at 37° C., 5% CO₂. Compounds were added as 1 μl ofa 1 mM DMSO stock solution for a final screening concentration of 10 μM,using low-volume tips for transfer (Molecular Bioproducts). Cells werethen incubated in the presence of compound for an additional sixteenhours, again at 37° C., 5% CO₂. Following incubation 70 μl of theculture medium was removed from each well to allow for reagent additionand plates were allowed to equilibrate to room temperature for thirtyminutes. Firefly luciferase reporter activity was then read by theaddition of thirty microliters of Dual Glo Luciferase reagent (Promega),followed by one hour incubation at room temperature to allow foradequate signal buildup. Luminescence counting was conducted on aMicrobeta Trilux using a 1 second read time. Renilla luciferase reporteractivity was measured following addition of 30 μl Stop and GloLuciferase reagent (Promega) and incubation identical to the one carriedout for the firefly luciferase one.

Compound scores were interpreted as firefly luciferase activity dividedby renilla luciferase activity, normalized to the plate's DMSO control.Using a preliminary screen of the NCI Diversity set as a guide (1990compounds), a hit threshold of three times the DMSO control readout waschosen to achieve a target hit rate of 1%; wells with this thresholdvalue typically fell three standard deviations from the plate mean. Alldata analysis was conducted using the BioAssay HTS software package(CambridgeSoft).

With this format, signal-to-noise and signal-to-background typically ranat 100 and 10 respectively, with thapsigargin (TG) an agent known toinduce eIF2α phosphorylation at 100 nM.

102,709 compounds in the NCI Open Chemical Repository were then screenedusing this HTS assay. Of these, approximately 1200 compounds wereidentified as hits in the primary screen (1.2% hit rate). Initial hitswere confirmed by repeating the same dual luciferase assay in 96 wellplates. Briefly, 20,000 cells/well were plated in triplicate for eachconcentration (10, 5 and 2.5 μM) of the compounds. The compounds thatincreased firefly/renilla luciferase ratio at least three-fold above thesame ratio in the DMSO treated wells were considered confirmed hits. Thefinal number of confirmed hits was 648 (See Table 5).

Lead Scaffolds

A review of the confirmed hits identified N,N′-diarylureas as aprivileged scaffold that can provide attractive leads. Usingcommercially available sources we assembled a 120 member lead findinglibrary of N,N′-diarylureas with various substitutions. Among thesecompounds three aryl-substituted active and one inactiveN,N′-diarylureas were selected for further evaluation (FIG. 7A). FIG. 7Bshows dose dependent effects of selected compounds on firefly/renillaluciferase ratio in KLN-tTA/pBISA-DL^((ATF-4)) cells.

Characterization of N,N′-Diarylurea Compounds in Secondary Assays

In order to validate the N,N′-diarylurea compounds bona fide modifiersof the abundance of the eIF2-GTP-Met-tRNA_(i) ternary complex, wedetermined effects selected active and inactive N,N′-diarylureas onendogenous cellular markers of the ternary complex. For example,reducing amount of the ternary complex increases translation of ATF-4,which results in elevated expression of CHOP mRNA and protein.Therefore, expression of endogenous CHOP mRNA and CHOP protein inKLN-tTA/pBISA-DL^((ATF-4)) cells were utilized as secondary assays tovalidate N,N′-diarylurea compounds as modifiers of theeIF2⋅GTP⋅Met-tRNA_(i) ternary complex. As shown in FIGS. 7C and D,active N,N′-diarylureas did indeed induce expression of CHOP mRNA andprotein. These findings demonstrate that the ternary complex assayreported herein is a very valuable tool for screening the chemicalrepositories for the inhibitors of translation initiation that reducethe amount of the ternary complex. To rule out the possibility that theactivity of N,N′-diarylurea compounds was confined to a single celltype, the effects of these compounds on the ternary complex assay andthe expression of CHOP mRNA was assayed in CRL-2351, PC-3, CRL-2813human breast, prostate, and melanoma cancer cell lines respectively. Forthe reporter gene assay, the three human cell lines were co-transfectedwith tTA expression vector and the pBISA-DL^((ATF-4)) dual luciferaseexpression vector shown in FIG. 6. As shown in FIGS. 8A-C, the activeN,N′-diarylureas displayed significant activity in all the cell linestested, albeit with different potencies. The expression of CHOP mRNA wasassayed by real time PCR in the same cell lines treated with 5 or 20 μMof each compound. As shown in FIG. 8D, the effect of all four compoundson CHOP mRNA expression closely followed their effect in thefirefly/renilla ratio.

The effect of certain compounds on ternary complex activity has beenstudied and the results presented below

Ternary Complex Activity Maximum effect (E_(max), Maximum effective doseCompound Structure fold over control) C_(max) (μM)

13 10

Inactive Not applicable

8.5 80

N′N′-Diarylurea Compounds Modify Availability of theeIF2-GTP-Met-tRNA_(i) Ternary Complex by Causing Phosphorylation ofeIF2α

The amount of the eIF2 GTP Met-tRNA_(i) complex can be reduced byphosphorylation of eIF2α, reduced expression of Met-tRNA_(i), or eIF2αphosphorylation independent reduction in the activity of eIF2B, the eIF2guanine nucleotide exchange factor. To determine which of these was thecase, the effect of active N,N′-diarylureas on the phosphorylation ofeIF2α was studied. KLN-tTA/pBISA-DL^((ATF-4)) or PC-3 prostate cancercells were incubated with three active and one inactive N,N′-diarylureacompounds and determined the phosphorylation of eIF2α by Western blotanalysis. As shown in FIG. 9A, three N,N′-diarylureas that increasedfirefly/renilla luciferase ratio and induce endogenous CHOP also causedthe phosphorylation of eIF2α, whereas the inactive N,N′-diarylureacompound had no effect on eIF2α phosphorylation. To determine if thephosphorylation of eIF2α was responsible for reduced amount of theternary complex in the cells treated with N,N′-diarylurea compounds,previously generated PC-3 human prostate cancer cell lines in whichexpression of endogenous eIF2α was replaced with recombinant wild type(WT) or non-phosphorylatable eIF2α-S51A were utilized. These cells wereco-transfected with tTA and pBISA-DL^((ATF-4)) dual luciferaseexpression vector and treated with three active and one inactiveN,N′-diarylurea compounds. FIG. 9B demonstrates replacement ofendogenous eIF2α with the non-phosphorylated eIF2α-S51A abrogated theactivity of N,N′-diarylureas in this assay. These finding demonstrateconclusively that N,N′-diarylurea compounds reduce the amount of theternary complex by causing phosphorylation of eIF2α.

N,N′-Diarylurea Compounds Reduce Expression of Cyclin D1

As shown in FIG. 11, active N,N′-diarylurea compounds inhibited cyclinD1 protein expression without any effect on p27^(Kip1) protein. Theseagents had no effect on the level of cyclin D1 mRNA indicating that theyinhibit cyclin D1 expression at the level of translation.

N,N′-Diarylurea Compounds Specifically Activate Heme Regulated Inhibitor(HRI)

Four distinct kinases are shown to specifically phosphorylate eIF2α inresponse to the metabolic state of the cells or external stimuli. Theseare PKR, PKR-like endoplasmic reticulum kinase (PERK), general controlderepressible kinase 2 (GCN2), and heme regulated inhibitor. Todetermine if N,N′-diarylurea compounds cause phosphorylation of eIF2α bycausing activation of one or more of these kinases, knockdown expressionof these kinases was assayed in KLN-tTA/pBISA-DL^((ATF-4)) cellsindividually or in combinations, treating the cells with compound #1781or DMSO. As seen in FIG. 12A, knocking down the expression of PKR, PERK,or GCN had no effect on the induction of F-luc/R-luc ratio by compound#1781. In contrast, knocking down the expression of HRI almostcompletely abrogated activity of the compound #1181 (FIG. 12A).Furthermore simultaneous knocking down of PKR, PERK, and GCN2 failed toabrogate effects of #1781 indicating that these three kinases do notplay a role in the induction of eIF2α phosphorylation by N,N′-diarylureacompounds. Furthermore real-time PCR analysis revealed that knockingdown HRI expression but not that of PKR, PRK or GCN2 abrogated inductionof CHOP mRNA by compound #1781, further supporting the finding that theHRI is the molecular target of N,N′-diarylurea compounds (FIG. 12B). Tofurther confirm these data, KLN-tTA/pBISA-DL^((ATF-4)) cells weretransfected with or without siRNA against HRI followed by theirtreatment with one inactive and three active N,N′-diarylurea compoundsor vehicle. As shown in FIG. 11C, knocking down the expression of HRIabrogated induction of R-luc/R-luc ratio (indicative of the limitedavailability of the ternary complex) by all three active compounds withno effect on the inactive compound (FIG. 11C). Finally, all four kinaseswere knocked down by about the same efficiency, ruling out thepossibility that the lack of effect by PKR, PERK, and GCN2 was due tofailure of siRNA knockdown (FIG. 11D). Taken together these data clearlydemonstrated that N,N′-diarylurea compounds cause phosphorylation ofeIF2α specifically by activating HRI.

Heme Regulated Inhibitor (HRI) Mediates Phosphorylation of eIF2α byN,N′-Diarylureas

Further studies were done to determine which eIF2α kinase(s) mediatephosphorylation of eIF2α by N,N′-diarylureas. In accordance with thisaspect, the expression of each one of the four eIF2α kinases was knockeddown either individually or in all possible combinations. MouseKLN-tTA/pBISA-DL^((ATF-4)) and human CRL-2813 melanoma cells weretransfected with siRNAs targeting PKR, GCN2, PERK or HRI, which knockeddown their respective mRNAs with 70-80% efficiency (see data presentedin Table 1).

TABLE 1 Efficiency of siRNAs in knocking down the expression of eIF2□kinase mRNAs in KLN-tTA/pBISA-DL^((ATF-4)) and CRL-2813 cancer cells.Knockdown efficiency (%) PERK GCN2 PKR HRI KLN 68.8 ± 3.5 75.3 ± 0.779.8 ± 5.6 65.7 ± 2.5 CRL-2813 82.5 ± 5.7 80.3 ± 1.5 69.4 ± 2.6 70.5 ±3.4

The co-transfected cells were treated with vehicle or an activeN,N′-diarylurea, compound #1781, and determined the normalizedF-luc/R-luc ratio. FIG. 13A shows that reduced expression of HRIsignificantly abrogated the activity of #1781. In sharp contrast,knocking down PKR, PERK, or GCN2 expression either individually or indouble or triple combination had no effect on the activity of #1781.Consistent with these results, silencing HRI but not the other eIF2αkinases abrogated the increased expression of CHOP mRNA induced bycompound #1781 (FIG. 13B). Furthermore, silencing of HRI reduced theinduction of eIF2α phosphorylation by #1781 (FIG. 13C). Finally, studiesin additional cell lines with N,N′-diarylureas showed that knocking-downexpression of HRI but not other eIF2 kinases abrogated the effect of allactive N,N′-diarylureas on the ternary complex abundance in these celllines (FIGS. 13D and 13E). Taken together, these data demonstrate thatactivation of HRI mediates the phosphorylation of eIF2α, the reducedavailability of the ternary complex and the other downstream effectsinduced by active N,N′-diarlyureas.

N,N′-Diarylureas Activate HRI in Cell-Free Lysates

Compound #1781 was added to lysates of CRL-2813 cells orheme-supplemented rabbit reticulocytes and phosphorylation of eIF2α byWestern blot was determined. As shown in FIG. 14, compound #1781 causedphosphorylation of eIF2α in cell lysates in a dose dependent mannerruling out the possibility the N,N′-diarylureas activate HRI due tocellular cytotoxicity.

N,N′-Diarylureas Inhibit Cell Proliferation by Reducing the Availabilityof the eIF2⋅GTP⋅Met-tRNA_(i) Ternary Complex

Reduced availability of the ternary complex causes inhibition oftranslation initiation and thereby of cell proliferation. Cellproliferation was selected as a biological response parameter todemonstrate target specificity and in vitro potency of N,N′-diarlyureas.The effects of N,N′-diarlyureas on the proliferation KLN mouse squamouscell carcinoma, CRL-2351 human breast, CRL-2813 human melanoma, A549human lung and PC-3 human prostate cancer cell lines were tested.N,N′-diarylureas active in the ternary complex assay were potentinhibitors of cells proliferation (see data presented in Table 2).

TABLE 2 Effect of N,N -diarylureas on proliferation of human cancercells IC₅₀ ^(*)(μM) Cell line 1527 1780 1781 KM094748 PC-3 8.3 0.9 1.11.4 KLN >20 14.8 17.1 8.5 CRL-2813 20 0.1 0.5 0.3 CRL-2351 9.5 1.3 3.00.1 A549 >20 0.8 1.2 1.3 *Concentration of compound that inhibit cellproliferation by 50%.

To determine if N,N′-diarylureas inhibit cell proliferation by reducingthe availability of the ternary complex, the effect of N,N′-diarylureason proliferation of previously described transgenic PC-3 human prostatecancer cell lines expressing either the non-phosphorylatable eIF2α-S51Amutant or the eIF2α-WT was studied. The results of these studies, shownin FIGS. 15A and 15B demonstrate that PC-3 cancer cells expressing thenon-phoshorylatable eIF2α-S51A mutant were resistant while thoseexpressing eIF2α-WT were sensitive to the inhibition of cellproliferation by N,N′-diarylureas. Reducing the expression of HRI, theeIF2α kinase that mediates N,N′-diarlyurea induced phosphorylation ofeIF2α similarly abrogates the effect of these agents on cellproliferation (FIGS. 15C and 15D). Taken together, these datademonstrate that N,N′-diarlyureas possess the required potency andspecificity to interrogate the role of the eIF2-GTP-Met-tRNA_(i) ternarycomplex in normal physiology and pathobiology of human disorders.

Expression of HRI Correlates with the Sensitivity of Cancer Cells toN,N′-Diarylureas

To determine correlate the sensitivity of the various cell lines toanti-proliferative effects of N,N′-diarylureas with the expression ofHRI, cell lysates were probed with anti-HRI antibodies and relativelevel of HRI expression was correlated with the inhibition of cellproliferation exerted by N,N′-diarylureas. KLN cells, which expressundetectable levels of HRI are very resistant to inhibition of cellproliferation by N,N′-diarylureas whereas CRL-2813 cells that expresshigh level of HRI are most sensitive (see Table 2 and FIG. 16).

N,N′-Diarylureas Display No Apparent Toxicity In Vivo

To study toxicity effects, mice were treated with various doses ofcompound #1781 or vehicle. As shown in FIG. 17, seven consecutive dayadministration of #1781 had no adverse effect on weight gain or foodconsumption of mice even at the highest dose tested indicating thatN,N′-diarylureas can be utilized to probe normal and patho-biology ofthe ternary complex in vivo.

Discussion

The ternary complex assay described herein is particularly robustbecause expression of both reporters is controlled by the sameenhancer/promoter complex, therefore any effect of the test compounds onthe transcription will be same for both reporters. Furthermore anyeffect of the test compounds on translation elongation or terminationwill be similar for both reporters. Because of these features, theternary complex assay described herein controls for many variables atonce. In addition, the primary assay is backed by the secondary assayssuch as the expression of CHOP protein and mRNA, which faithfullyreflects the abundance of the ternary complex. The specificity of theassay was further demonstrated by testing well-known anti-cancer agentsfor their effects on the abundance of the ternary complex. None of theseanti-cancer agents with no known effect on the formation of the ternarycomplex showed any activity, indicating that this assay is suitable foridentification of mechanism specific active compounds. In addition toidentifying privileged scaffolds that could be utilized for design offocused libraries for lead generation, a library of N,N′-diarylureas wasprepared and studied using the ternary complex assay. Furthercharacterization of selected compounds indicated that N,N′-diarylureacompounds reduced the amount of the ternary complex by causingphosphorylation of eIF2α. These findings indicate that the ternarycomplex assay is highly suitable for guiding development of translationinitiation inhibitors, and that these N,N′-diarylurea compounds do infact display potent anti-proliferative activity correlated with theiractivity in the ternary complex assay.

The data described herein indicate that the active N,N′-diarylureacompounds cause phosphorylation of eIF2α, induce expression of CHOP mRNAand protein and potently inhibit cell proliferation. The N,N′-diarylureacompounds also preferentially inhibited expression of cyclin D1. Thedata presented herein demonstrate that phosphorylation of eIF2α byN,N′-diarylurea compounds is required for reducing amount of the ternarycomplex by these agents. It was further demonstrated thatN,N′-diarylureas compounds inhibit cell proliferation by causingphosphorylation of eIF2α with IC₅₀ values in the low/sub-micromolarrange.

The data presented herein demonstrates the clear potential for targetingof the eIF2-GTP-Met-tRNA_(i) ternary complex in a cell based highthroughput screening campaign to develop translation initiationinhibitors and potential of N,N′-diarylurea compounds for development ofnovel mechanism specific agents for cancer therapy.

Materials and Methods

Cell Growth Assay: Cell growth was measured by the SRB assay.

Plasmids: The bi-directional mammalian expression vector pBI (Clontech,CA) was modified to expand the multiple cloning sites MCSs andthereafter named pBISA. This vector contains seven copies of thetetracycline regulated transactivator response element (TRE), whichtogether act as core promoter/enhancer. The TRE is flanked on both sidesby minimal human cytomegalovirus (CMV) minimal promoters allowingbi-directional transcription and two (MCS). Firefly and renillaluciferases were subcloned into MCS-I and MCS-II, respectively. Thisbase plasmid, designated pBISA-DL, transcribes two mRNAs that containthe 90 nucleotide plasmid derived 5′ UTR (same sequence in both mRNAs),and the ORF encoding either firefly or renilla luciferase followed by apolyadenylation sequence. This plasmid was further modified by insertingthe 5′ UTR of ATF-4 into MCS-I in front of the firefly luciferase mRNA.Transcription from this direction generates an mRNA that contains thefirefly luciferase ORF preceded by a 5′ UTR composed of 90 nucleotidesderived from the plasmid and 267 nucleotides derived from the 5′ UTR ofATF-4 mRNA. Transcription from the other direction generates an mRNAthat contains the renilla luciferase ORF preceded only by the90-nucleotide plasmid-derived sequence in the 5′ UTR (FIG. 1B). Thisexpression plasmid is called pBISA-DL^((ATF-4)).

Stable and transient transfection: Cells were seeded at the density of10⁵ in 60-mm (stable transfection) or 10⁴ cells per well of 96-wellplate (transient transfection) plates and transfected one day laterusing the Qiagen Transfectamine transfection kit. For selection ofstable cell lines, transfected cells were transferred to 100-mm platesand selected with appropriate antibiotics.

Western blotting: Cell extracts were separated by SDS-PAGE and probedwith anti-phosphoserine-51-eIF2α (PS51-eIF2α), anti-total eIF2α-specificantibodies (PS51-eIF2α (Biosource International, Hopkinton, Mass.),anti-CHOP, or anti β-actin (Santa Cruz Biotechnology, CA) as described.

Robotics: Liquid handling was conducted on a Biomek FX (BeckmanCoulter). Luminescence measurements were conducted on a Microbeta Trilux(Perkin Elmer). Both are components on a Sagian Core robotic platform(Beckman Coulter).

Dual luciferase assay: Cells or minced tumors expressing firefly andrenilla luciferases were lysed and the extracts assayed with a glow typedual luciferase assay kit, per manufacturer's instruction (Promega Inc.,Madison, Wis.).

Real time PCR: For real time PCR, total RNA was extracted with TaqManGene Expression Cells-to-Ct™ Kit (Applied Biosystems, Branchburg, N.J.)according to manufacturer's protocol. Contaminating DNA was removed byDNase I treatment. 1-Step Real-time PCR was performed on a Bio-RadiCycler IQ5 system by using B-R 1-Step SYBR Green qRT-PCR Kit (QuantaBioSciences, Gaithersburg, Md.) according to manufacturer'sspecifications. The thermal cycler conditions were as follows: 10minutes at 50° C., hold for 5 minutes at 95° C., followed by 2-step PCRfor 45 cycles of 95° C. for 15 seconds followed by 60° C. for 30seconds. All PCRs were performed triplicate in independent PCR runs.Mean values of these repeated measurements were used for calculation. Tocalibrate the results, all the transcripts quantities were normalized to18S rRNA (was 18S ribosomal RNA-like mRNA in mouse). The followingprimers were used in real-time PCR reactions:

Human CHOP (SEQ ID NO: 1) 5′ AGAACCAGGAAACGGAAACAGA 3′ (SEQ ID NO: 2) 5′TCTCCTTCATGCGCTGCTTT 3′ Mouse CHOP (SEQ ID NO: 3) 5′CATACACCACCACACCTGAAAG 3′ (SEQ ID NO: 4) 5′ CCGTTTCCTAGTTCTTCCTTGC 3′Human Cyclin D1 (SEQ ID NO: 5) 5′ CGGAGGAGAACAAACAGA 3′ (SEQ ID NO: 6)5′ TGAGGCGGTAGTAGGACA 3′ Mouse Cyclin D1 (SEQ ID NO: 7) 5′TACCGCACAACGCACTTTCTT 3′ (SEQ ID NO: 8) 5′ CGCAGGCTTGACTCCAGAAG 3′Human/Mouse 18s rRNA (SEQ ID NO: 9) 5′ CGGCGACGACCCATTCGAAC 3′(SEQ ID NO: 10) 5′ GAATCGAACCCTGATTCCCCGTC 3′

Example II Structure-Activity Relationship (SAR) Study ofN,N′-Diarylureas as Inhibitors of Translation Initiation, PotentAnti-Cancer Agents

The general synthetic approaches to produce N,N′-diarylurea compounds ofthe present invention are set forth below.

Chemistry

The first series of molecules in this example, most of which aresymmetrical N,N′-diarylureas substituted by heteroatoms or groups ofheteroatoms, was prepared by using appropriate commercially availablearyl isocyanates and aryl amines according to Scheme 1.

-   -   Reagents and conditions: (i) anilines, 1,4-dioxane, 55° C.

The synthesis of compounds 1-3 was carried out in one step in1,4-dioxane at 55° C. overnight. The same simple procedure using5-aminocresol or 3-methoxy-4-methylaniline as new starting aryl amineswas followed for the elaboration of the unsymmetrical N,N′-diarylureas4-9 according to Scheme 2.

-   -   Reagents and conditions: (i) anilines, 1,4-dioxane, 55° C.

Analogs 11-13 and 15-17 were prepared in a slightly different manner.The synthesis began by the elaboration of two different substitutedanilines starting from 2-methyl-5-nitrophenol. Compound 10, which wasthe precursor of N,N′-diarylureas 11-13, was obtained via a classicMitsunobu coupling reaction in presence of N,N-dimethylethanolamineaccording to Scheme 3.

-   -   Reagents and conditions: (i) N,N-dimethylethanolamine, PPh₃,        DEAD, THF, 0° C.; (ii) SnCl₂, EtOH, 90° C.; (iii)        phenylisocyanates, dioxane, 55° C.

In the same way, compound 14, which was the precursor ofN,N′-diarylureas 15-17 was obtained starting from4-(2-hydroxymethyl)morpholine according to Scheme 4.

-   -   Reagents and conditions: (i) 4-(2-hydroxyethyl)morpholine, PPh₃,        DEAD, THF, 0° C.; (ii) SnCl₂, EtOH, 90° C.; (iii)        phenylisocyanates, dioxane, 55° C.

After reduction of the nitro group in amine by the use of tin chloridein ethanol at 90° C., the substituted anilines were directly coupled tothe same various isocyanates in 1,4-dioxane at 55° C. overnight toproduce 11-13 and 15-17.

In order to couple piperazine with 2-methyl-5-nitrophenol via aMitsunobu reaction (compound 19), the secondary amine was firstprotected by a benzyloxycarbonyl group. The protection was carried outwith benzylclhoroformate and a solution of NaOH 4N to afford 18according to Scheme 5.

-   -   Reagents and conditions: (i) benzylchloroformate, NaOH 4N,        CH₃CN/H₂O; (ii) 2-methyl-5-nitrophenol, PPh₃, DEAD, THF, 0°        C.; (iii) SnCl₂, EtOH, 90° C.; (iv) phenylisocyanates, dioxane,        55° C.; (v) H₂, Pd—C, MeOH, 1 atm; (vi) HCl 4N, dioxane.

After the coupling reaction using triphenylphosphine and DEAD in THF,the nitro group was, as previously described, reduced in amine andcoupled to the same various isocyanates to produce protectedintermediates 20-22. Finally, after a hydrogenolysis carried out atatmospheric pressure under hydrogen and in presence of palladium oncarbon, a precipitation in a solution of HCl 4N in 1,4-dioxane allowedthe isolation of N,N′-diarylureas 23-25 as salts.

The last series of molecules in this example, in which heteroatoms wereincluded in the aromatic ring, was prepared starting from severalsubstituted pyridine and pyrimidine and using the same general procedurein 1,4-dioxane at 55° C. overnight according to Scheme 6.

-   -   Reagents and conditions: (i) 2-amino-3-hydroxypyridine, dioxane        55° C.

While compounds 26 and 27 appeared to be easily isolable bycrystallization or purification by preparative HPLC, compound 28, whichwas derivated from 2-amino-4-hydroxy-6-methylpyrimidine, appeared to benot soluble in any solvent. Because it could not be purified, thiscompound was removed from the structure-activity relationship (SAR)study.

General Procedure A for the Synthesis of Compounds 1-91,3-bis(3,4-dichlorophenyl)urea (Compound 1)

As a non-limiting example, 3,4-dichlorophenylisocyanate (188 mg, 1 mmol)and 3,4-dichloroaniline (178 mg, 1.1 mmol) were dissolved in 10 mL ofanhydrous dioxane. The reaction mixture was warmed to 55° C., stirredunder nitrogen over night and then cooled to room temperature. Thesolvent was removed under vacuum and the crude was purified twice bycrystallization in ethyl acetate/hexane to afford 1 (262 mg, 75%) as awhite powder.

1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]urea (Compound 2)

As another non-limiting example,4-chloro-3(trifluoromethyl)phenylisocyanate (222 mg, 1 mmol) and4-chloro-3-(trifluoromethyl)aniline (215 mg, 1.1 mmol) were usedfollowing the general procedure A to isolate 2 (250 mg, 60%) as a whitepowder.

1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea (Compound 3)

As another non-limiting example,3,5-bis(trifluoromethyl)phenylisocyanate (600 mg, 2.353 mmol) and3,5-bis(trifluoromethyl)aniline (647 mg, 2.824 mmol) were used followingthe general procedure A to isolate 3 (1140 mg, 78%) as a white powder.¹H NMR (500 MHz, CD₃OD, δ): 8.12 (s, 1H, CH_(arom.)), 7.59 (s, 1H,CH_(arom.)). ¹³C NMR (400 MHz, CD₃OD, δ): 152.89, 141.32, 132.55,131.80, 124.9, 122.2, 118.4, 115.24.

3-(3,4-dichlorophenyl)-1-(3-hydroxy-4-methylphenyl)urea (Compound 4)

As another non-limiting example, 3,4dichlorophenylisocyanate (202 mg,1.073 mmol) and 5-aminocresol (120 mg, 0.976 mmol) were used followingthe general procedure A to isolate 4 (244 mg, 81%) as a white powder. ¹HNMR (500 MHz, DMSO_(d6), δ): 9.24 (s, 1H, OH), 8.82 (s, 1H, NH), 8.57(s, 1H, NH), 7.85 (s, 1H, CH_(arom.)), 7.47 (d, J=11 Hz, 1H,CH_(arom.)), 7.28 (d, J=11 Hz, 1H, CH_(arom.)), 7.04 (s, 1H,CH_(arom.)), 6.90 (d, J=10 Hz, 1H, CH_(arom.)), 6.69 (d, J=10 Hz, 1H,CH_(arom.)), 2.02 (s, 3H, CH₃). ¹³C NMR (400 MHz, DMSO_(d6), δ): 156.09,152.84, 140.77, 138.45, 131.68, 131.19, 131.06, 123.54, 119.78, 118.84,118.35, 109.71, 105.96, 16.09.

3-[4-chloro-3-(trifluoromethyl)phenyl]-1-(3-hydroxy-4-methylphenyl)urea(Compound 5)

As another non-limiting example,4-chloro-3-(trifluoromethyl)phenylisocyanate (198 mg, 0.894 mmol) and5-aminocresol (100 mg, 0.813 mmol) were used following the generalprocedure A to isolate 5 (102 mg, 46%) as a white powder. ¹H NMR (500MHz, DMSO_(d6), δ): 9.26 (s, 1H, OH), 9.03 (s, 1H, NH), 8.64 (s, 1H,NH), 8.12 (s, 1H, CH_(arom.)), 7.58 (m, 2H, CH_(arom.)), 7.10 (s, 1H,CH_(arom.)), 6.92 (d, J=8 Hz, 1H, CH_(arom.)), 6.69 (d, J=8 Hz, 1H,CH_(arom.)), 2.04 (s, 3H, CH₃). ¹³C NMR (400 MHz, DMSO_(d6), δ): 156.09,125.93, 140.17, 138.38, 132.62, 131.05, 123.53, 122.72, 118.40, 109.79,106.04, 16.07.

3-[3,5-bis(trifluoromethyl)phenyl]-1-(3-hydroxy-4-methylphenyl)urea(Compound 6)

As another non-limiting example, 3,5-bis(trifluoromethyl)phenylisocyanate (200 mg, 0.784 mmol) and 5-aminocresol (106 mg, 0.862mmol) were used following the general procedure A to synthesize 9. Atthe end of the reaction, the crude was purified by flash chromatography(15 to 30% of ethyl acetate in cyclohexane). After concentration of thepure fractions, the white solid was crystallized in hexane to afford 6(260 mg, 52.5%) as a white powder. ¹H NMR (500 MHz, DMSO_(d6), δ): 9.27(s, 1H, OH), 9.25 (s, 1H, NH), 8.79 (s, 1H, NH), 8.11 (s, 2H,CH_(arom.)), 7.61 (s, 1H, CH_(arom.)), 7.12 (s, 1H, CH_(arom.)), 6.94(d, J=8 Hz, 1H, CH_(arom.)), 6.72 (d, J=8 Hz, 1H, CH_(arom.)), 2.05 (s,3H, CH₃). ¹³C NMR (500 MHz, DMSO_(d6), δ): 156.11, 152.97, 142.26,138.20, 131.48, 131.23, 130.97, 127.27, 125.10, 122.93, 118.76, 114.85,110.05, 106.30, 16.09.

Compound 7

As another non-limiting example, 3,4-dichlorophenylisocyanate (151 mg,0.803 mmol) and 3-methoxy-4-methylaniline (100 mg, 0.730 mmol) were usedfollowing the general procedure A to isolate 7 (204 mg, 86%) as a whitepowder. ¹H NMR (500 MHz, DMSO_(d6), δ): 8.91 (s, 1H, NH), 8.70 (s, 1H,NH), 7.88 (s, 1H, CH_(arom.)), 7.50 (d, J=9 Hz, 1H, CH_(arom.)), 7.30(d, J=9 Hz, 1H, CH_(arom.)), 7.19 (s, 1H, CH_(arom.)), 7.01 (d, J=8 Hz,1H, CH_(arom.)), 6.82 (d, J=8 Hz, 1H, CH_(arom.)), 3.76 (s, 3H, OCH₃),2.07 (s, 3H, CH₃). ¹³C NMR (400 MHz, DMSO_(d6), δ): 157.99, 152.97,140.71, 139.02, 131.70, 131.22, 130.87, 123.67, 119.91, 119.84, 119.00,110.72, 102.14, 55.72, 16.15.

Compound 8

As another non-limiting example,4-chloro-3-(trifluoromethyl)phenylisocyanate (151 mg, 0.682 mmol) and3-methoxy-4-methylaniline (85 mg, 0.620 mmol) were used following thegeneral procedure A to isolate 8 (102 mg, 46%) as a white powder. 1H NMR(500 MHz, DMSO_(d6), δ): 9.07 (s, 1H, NH), 8.74 (s, 1H, NH), 8.07 (s,1H, CH arom.), 7.60 (m, 2H, CH arom.), 7.17 (s, 1H, CH arom.), 7.00 (d,J=10 Hz, 1H, CH arom.), 6.82 (d, J=10 Hz, 1H, CH arom.), 3.74 (s, 3H,OCH₃), 2.06 (s, 3H, CH₃). 13C NMR (400 MHz, DMSO_(d6), δ): 157.99,153.05, 140.10, 138.95, 132.64, 130.87, 123.70, 119.95, 117.39, 110.85,102.22, 55.70, 16.14.

Compound 9

As another non-limiting example,3,5-bis(trifluoromethyl)phenylisocyanate (143 mg, 0.562 mmol) and3-methoxy-4-methylaniline (70 mg, 0.511 mmol) were used following thegeneral procedure A to isolate 9 (92 mg, 46%) as a white powder. ¹H NMR(500 MHz, DMSO_(d6), δ): 9.32 (s, 1H, NH), 8.90 (s, 1H, NH), 8.10 (m,2H, CH_(arom.)), 7.61 (s, 1H, CH_(arom.)), 7.18 (s, 1H, CH_(arom.)),7.00 (d, J=10 Hz, 1H, CH_(arom.)), 6.85 (d, J=10 Hz, 1H, CH_(arom.)),3.74 (s, 3H, OCH₃), 2.06 (s, 3H, CH₃). ¹³C NMR (400 MHz, DMSO_(d6), δ):157.98, 153.05, 142.59, 138.74, 131.50, 131.18, 130.86, 128.06, 125.35,122.64, 120.16, 118.59, 114.92, 111.08, 102.42, 55.73, 16.15.

General Procedure B (for the Synthesis of Compounds 10, 14 and 19)Compound 10

As another non-limiting example, 2-methyl-5-nitrophenol (2.00 g, 13.07mmol), N,N-dimethylethanolamine (1.31 mL, 13.07 mmol) andtriphenylphosphine (4.46 g, 16.99 mmol) were placed in a 100 mLround-bottomed flask under nitrogen. 40 mL of anhydrous THF were addedvia syringe at 0° C. After stirring the reaction mixture at thistemperature for 10 minutes, 7.32 mL of a solution ofdiethylazodicarboxylate 40% in toluene (2.93 g, 16.99 mmol) were addedvia syringe. The reaction was warmed to room temperature and stirredunder nitrogen for two hours. The solvents were removed under vacuum.Triphenylphosphine oxide formed during the reaction was precipitated ina mixture of ethyl acetate/hexane and filtrated. The crude was thenpurified by flash chromatography (0 to 2% of MeOH in DCM) to afford 10(2.11 g, 70%) as a yellow oil.

General Procedure C (for the Synthesis of Compounds 11-13, 14-16 and20-22)3-(3,4-dichlorophenyl)-1-{3-[2-(dimethylamino)ethoxy]-4-methylphenyl}urea(Compound 11)

As another non-limiting example, Compound 10 (262 mg, 1.169 mmol) wasdissolved in 10 mL of EtOH. SnCl₂H₂O (1316 mg, 5.848 mmol) was added andthe temperature was increased to 90° C. The reaction mixture was stirredfor 1.5 hours, cooled to room temperature and poured into iced water.The solution was made alkaline with solid NaOH and then extracted withDCM (3×30 mL). Organic extracts were combined, washed with water (60 mL)and brine (60 mL), dried over sodium sulfate, concentrated and finallydried under high vacuum over night to afford the substituted aniline(202 mg, 1.041 mmol) as a light-yellow oil. This compound was thendissolved in 10 mL of anhydrous dioxane and 3,4-dichlorophenylisocyanate(254 mg, 1.353 mmol) was added. The reaction mixture was warmed to 55°C., stirred under nitrogen overnight and then cooled to roomtemperature. The crude was purified by flash chromatography (0 to 2% ofMeOH in DCM). After concentration of the pure fractions, the obtainedwhite solid was crystallized in hexane to afford 11 (260 mg, 58%) as awhite powder. ¹H NMR (300 MHz, DMSO_(d6), δ): 8.92 (s, 1H, NH), 8.69 (s,1H, NH), 7.85 (s, 1H, CH_(arom.)), 7.45 (d, J=8.7 Hz, 1H, CH_(arom.)),7.27 (d, J=8.7 Hz, 1H, CH_(arom.)), 7.15 (m, 1H, CH_(arom.)), 6.98 (d,J=7.8 Hz, 1H, CH_(arom.)), 6.80 (d, J=7.8 Hz, 1H, CH_(arom.)), 3.98 (t,J=5.7 Hz, 2H, OCH₂), 2.63 (t, J=5.7 Hz, 2H, CH₂N), 2.21 (s, 6H,N(CH₃)₂), 2.04 (s, 3H, CH₃). ¹³C NMR (400 MHz, DMSO_(d6), δ): 157.22,152.97, 140.67, 138.88, 131.68, 131.19, 130.90, 123.66, 120.11, 119.89,118.99, 110.88, 103.09, 66.72, 58.30, 46.30, 16.11.

3-[4-chloro-3-(trifluoromethyl)phenyl]-1-{3-[2-(dimethylamino)ethoxy]-4-methylphenyl}urea(Compound 12)

As another non-limiting example, Compound 10 (155 mg, 0.692 mmol) and4-chloro-3-(trifluoromethyl) phenyl isocyanate (151 mg, 0.682 mmol) wereused following the general procedure B to synthesize 12. At the end ofthe reaction, the mixture was precipitated in a solution of HCl 4N indioxane. After filtration, the white solid was dissolved in acetic acidand purified by preparative HPLC (10 to 40% of acetonitrile in waterwith 0.1% of acetic acid) to afford 12 (161 mg, 52%) as a white powder.¹H NMR (500 MHz, DMSO_(d6), δ): 9.26 (s, 1H, NH), 8.86 (s, 1H, NH), 8.09(s, 1H, CH_(arom.)), 7.62 (m, 2H, CH_(arom.)), 7.18 (s, 1H, CH_(arom.)),7.15 (d, J=8 Hz, 1H, CH_(arom.)), 6.85 (d, J=8 Hz, 1H, CH_(arom.)), 4.02(t, J=5.5 Hz, 2H, OCH₂), 2.71 (t, J=5.5 Hz, 2H, CH₂N), 2.27 (s, 6H,N(CH₃)₂), 2.08 (s, 3H, CH₃).

3-[3,5-bis(trifluoromethyl)phenyl]-1-{3-[2-(dimethylamino)ethoxy]-4-methylphenyl}urea(Compound 13)

As another non-limiting example, Compound 10 (248 mg, 1.107 mmol) and3,5-bis(trifluoromethyl) phenylisocyanate (326 mg, 1.280 mmol) were usedfollowing the general procedure B to synthesize 13. At the end of thereaction, the crude was purified by flash chromatography (2 to 8% ofMeOH in DCM). After concentration of the pure fractions, the white solidwas crystallized in hexane to afford 13 (260 mg, 52.5%) as a whitepowder. ¹H NMR (500 MHz, DMSO_(d6), δ): 9.37 (s, 1H, NH), 8.90 (s, 1H,NH), 8.12 (s, 2H, CH_(arom.)), 7.62 (m, 1H, CH_(arom.)), 7.19 (s, 1H,CH_(arom.)), 7.03 (d, J=8 Hz, 1H, CH_(arom.)), 6.88 (d, J=8 Hz, 1H,CH_(arom.)), 4.03 (t, J=5.5 Hz, 2H, OCH₂), 2.68 (t, J=5.5 Hz, 2H, CH₂N),2.25 (s, 6H, N(CH₃)₂), 2.09 (s, 3H, CH₃). ¹³C NMR (400 MHz, DMSO_(d6),δ): 157.24, 153.05, 142.58, 138.61, 131.51, 131.19, 130.90, 127.78,125.34, 122.62, 120.46, 118.53, 114.89, 111.26, 103.40, 66.77, 58.30,46.28, 15.95.

Compound 14

As another non-limiting example, 2-methyl-5-nitrophenol (2.00 g, 13.07mmol), 4(2-hydroxyethyl)morpholine (1.71 mg, 13.07 mmol),triphenylphosphine (4.46 g, 16.99 mmol) and 7.32 mL of a solution ofdiethylazodicarboxylate 40% in toluene (2.93 g, 16.99 mmol) were usedfollowing the general procedure B to synthesize 14. After treatments,the crude was purified by flash chromatography (0 to 3% of MeOH in DCM)to afford 14 (0.85 g, 23%) as a brown oil.

3-(3,4-dichlorophenyl)-1-{4-methyl-3-[2-(morpholin-4-yl)ethoxy]phenyl}urea(Compound 15)

As another non-limiting example, Compound 14 (278 mg, 1.045 mmol) and3,4-dichlorophenylisocyanate (285 mg, 1.118 mmol) were used followingthe general procedure C to synthesize 14. At the end of the reaction,the crude was purified by flash chromatography in normal phase (10 to 0%of cyclohexane in ethyl acetate). After concentration of the purefractions, the obtained white solid was crystallized in hexane to afford15 (217 mg, 49%) as a white powder. ¹H NMR (500 MHz, DMSO_(d6), δ): 8.94(s, 1H, NH), 8.70 (s, 1H, NH), 7.89 (s, 1H, CH_(arom.)), 7.51 (d, J=8.5Hz, 1H, CH_(arom.)), 7.32 (d, J=8.5 Hz, 1H, CH_(arom.)), 7.20 (s, 1H,CH_(arom.)), 7.02, (d, J=8 Hz, 1H, CH_(arom.)), 6.81 (d, J=8 Hz, 1H,CH_(arom.)), 4.05 (t, J=5.5 Hz, 2H, OCH₂), 3.58 (m, 4H, CH₂OCH₂), 2.73(t, J=5.5 Hz, 2H, CH₂N), 2.50 (m, 4H, N(CH₂)₂), 2.08 (s, 3H, CH₃). ¹³CNMR (400 MHz, DMSO_(d6), δ): 157.19, 152.96, 140.66, 138.88, 131.68,131.19, 130.91, 123.67, 120.15, 119.89, 118.98, 110.95, 103.22, 66.87,66.46, 57.62, 54.62, 15.98.

3-[4-chloro-3-(trifluoromethyl)phenyl]-1-{4-methyl-3-[2-(morpholin-4-yl)ethoxy]phenyl}urea(Compound 16)

As another non-limiting example, Compound 14 (267 mg, 1.004 mmol) and4-chloro-3-(trifluoromethyl) phenylisocyanate (241 mg, 1.091 mmol) wereused following the general procedure C to synthesize 16. At the end ofthe reaction, the crude was purified by flash chromatography (10 to 0%of cyclohexane in ethyl acetate). After concentration of the purefractions, the obtained white solid was crystallized in hexane to afford16 (263 mg, 58%) as a white powder. ¹H NMR (500 MHz, DMSO_(d6), δ): 9.11(s, 1H, NH), 8.74 (s, 1H, NH), 8.09 (s, 1H, CH_(arom.)), 7.61 (m, 2H,CH_(arom.)), 7.20 (s, 1H, CH_(arom.)), 7.02 (d, J=8 Hz, 1H, CH_(arom.)),6.83 (d, J=8 Hz, 1H, CH_(arom.)), 4.05 (t, J=5.5 Hz, 2H, OCH₂), 3.58 (m,4H, CH₂OCH₂), 2.73 (t, J=5.5 Hz, 2H, CH₂N), 2.50 (m, 4H, N(CH₂)₂), 2.08(s, 3H, CH₃). ¹³C NMR (400 MHz, DMSO_(d6), δ): 157.23, 153.07, 140.11,138.85, 1321.65, 130.92, 123.71, 120.27, 117.35, 110.12, 103.40, 66.77,66.37, 57.65, 54.28, 15.98.

3-[3,5-bis(trifluoromethyl)phenyl]-1-{4-methyl-3-[2-(morpholin-4-yl)ethoxy]phenyl}urea(Compound 17)

As another non-limiting example, Compound 14 (273 mg, 1.026 mmol) and3,5-bis(trifluoromethyl) phenylisocyanate (285 mg, 1.118 mmol) were usedfollowing the general procedure C to synthesize 17. At the end of thereaction, the crude was purified by flash chromatography in normal phase(20 to 10% of cyclohexane in ethyl acetate). After concentration of thepure fractions, the obtained white solid was crystallized in hexane toafford 17 (235 mg, 48%) as a white powder. ¹H NMR (500 MHz, DMSO_(d6),δ): 10.11 (s, 1H, NH), 9.34 (s, 1H, NH), 8.11 (s, 2H, CH_(arom.)), 7.60(s, 1H, CH_(arom.)), 7.22, (s, 1H, CH_(arom.)), 7.02 (d, J=7.5 Hz, 1H,CH_(arom.)), 6.85 (d, J=7.5 Hz, 1H, CH_(arom.)), 4.05 (m, 2H, OCH₂),3.58 (m, 4H, CH₂OCH₂), 2.73 (m, 2H, CH₂N), 2.51 (m, 4H, N(CH₂)₂), 2.08(s, 3H, CH₃).

Compound 18

As another non-limiting example, piperazine (3.00 g, 23.04 mmol) wasdissolved in 15 mL of water in a three-neck round-bottomed flask. Asolution of benzylchloroformate (3.95 mL, 27.65 mmol) in 15 mL ofacetonitrile was added drop wise via isobar cylindrical funnel. In orderto maintain the pH around 9, a solution of NaOH 4N was added drop wisevia a second isobar cylindrical funnel. The reaction mixture was stirredover night at room temperature and then extracted with DCM (2×75 mL).The aqueous phase containing the final compound was acidified with HCl3N and extracted with DCM (3×75 mL). Organic extracts were combined,washed with brine (150 mL), dried over sodium sulfate and concentratedunder vacuum. The crude was purified by flash chromatography (0 to 2% ofMeOH in DCM) to afford 18 (5.41 g, 90%) as a colorless oil.

Compound 19:

As another non-limiting example, 2-methyl-5-nitrophenol (1.70 g, 11.11mmol), compound 18 (2.94 mg, 11.1 mmol), triphenylphosphine (3.79 g,14.44 mmol) and 6.27 mL of a solution of diethylazodicarboxylate 40% intoluene (2.51 g, 14.44 mmol) were used following the general procedure Bto synthesize 19. After treatments, the crude was then purified by flashchromatography (0 to 3% of MeOH in DCM) to afford 19 (3.82 g, 86%) as ayellow oil.

Compound 20

As another non-limiting example, Compound 19 (1.172 g, 2.937 mmol) and3,4-dichlorophenylisocyanate (0.545 g, 2.778 mmol) were used followingthe general procedure C to synthesize 20. After treatments, the crudewas purified by flash chromatography (40 to 0% of cyclohexane in ethylacetate) to afford 20 (0.984 g, 60%) as a white powder.

Compound 21

As another non-limiting example, Compound 19 (1.042 g, 2.609 mmol) and4-chloro-3-(trifluoromethyl) phenylisocyanate (0.545 g, 2.459 mmol) wereused following the general procedure C to synthesize 21. Aftertreatments, the crude was purified by flash chromatography (40 to 0% ofcyclohexane in ethyl acetate) to afford 21 (1.045 g, 68%) as a whitepowder.

Compound 22

As another non-limiting example, Compound 19 (992 mg, 2.486 mmol) and3,5-bis(trifluoromethyl) phenylisocyanate (600 mg, 2.352 mmol) were usedfollowing the general procedure C to synthesize 22. After treatments,the crude was purified by flash chromatography (5 to 0% of cyclohexanein ethyl acetate) to afford 22 (945 mg, 71%) as a white powder.

General Procedure D (for the Synthesis of Compounds 23-25)4-[2-(5-{[(3,4-dichlorophenyl)carbamoyl]amino}-2-methylphenoxy)ethyl]piperazine-1,4-diiumdichloride (Compound 23)

As another non-limiting example, Compound 20 (870 mg, 1.561 mmol) wasdissolved in 20 mL of MeOH and Pd—C (10% by weight, 93 mg) was carefullyadded. The reaction mixture was stirred under a flux of hydrogen for 2hours at atmospheric pressure and room temperature (the reaction wasmonitored by LCMS) and then filtered through a pad of celite. Thefiltrate was concentrated and purified by HPLC (10 to 45% ofacetonitrile in water with 0.1% of acetic acid). After concentration ofthe pure fractions, the compound was precipitated in a solution of HCl4N in dioxane to afford 23 (379 mg, 49%) as a white powder. ¹H NMR (500MHz, DMSO_(d6), δ): 12.03 (s, 1H, NH), 9.77 (s, 1H, NH), 9.57 (m, 2H,NH), 9.36 (s, 1H, NH), 7.89 (s, 1H, CH_(arom.)), 7.50 (d, J=8.5 Hz, 1H,CH_(arom.)), 7.32 (m, 2H, CH_(arom.)), 7.05 (d, J=8 Hz, 1H, CH_(arom.)),6.83 (d, J=8 Hz, 1H, CH_(arom.)), 4.33 (m, 2H, OCH₂), 3.74-3.39 (m, 10H,CH₂N), 2.13 (s, 3H, CH₃).

4-{2-[5-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-2-ethylphenoxy]ethyl}piperazine-1,4-diiumdichloride (Compound 24)

As another non-limiting example, Compound 21 (930 mg, 1.573 mmol) wastreated following the general procedure D and purified by HPLC (15 to45% of acetonitrile in water with 0.1% of acetic acid). Afterconcentration of the pure fractions, the compound was precipitated in asolution of HCl 4N in dioxane to afford 24 (600 mg, 72%) as a whitepowder. ¹H NMR (500 MHz, DMSO_(d6), δ): 12.00 (s, 1H, NH), 9.94 (s, 1H,NH), 9.61 (m, 2H, NH), 9.40 (s, 1H, NH), 8.11 (s, 1H, CH_(arom.)), 7.61(m, 2H, CH_(arom.)), 7.29 (s, 1H, CH_(arom.)), 7.06 (d, J=8 Hz, 1H,CH_(arom.)), 6.85 (d, J=8 Hz, 1H, CH_(arom.)), 4.37 (m, 2H, OCH₂),3.85-3.46 (m, 10H, CH₂N), 2.13 (s, 3H, CH₃).

4-{2-[5-({[3,5-bis(trifluoromethyl)phenyl]carbamoyl}amino)-2-methylphenoxy]ethyl}piperazine-1,4-diiumdichloride (Compound 25)

As another non-limiting example, Compound 22 (840 mg, 1.345 mmol) wastreated following the general procedure D and purified by HPLC (15 to45% of acetonitrile in water with 0.1% of acetic acid). Afterconcentration of the pure fractions, the compound was precipitated in asolution of HCl 4N in dioxane to afford 25 (318 mg, 42%) as a whitepowder. ¹H NMR (500 MHz, DMSO_(d6), δ): 12.13 (s, 1H, NH), 10.45 (s, 1H,NH), 9.71 (m, 2H, NH), 9.58 (s, 1H, NH), 8.10 (s, 2H, CH_(arom.)), 7.61(m, 12H, CH_(arom.)), 7.29 (s, 1H, CH_(arom.)), 7.07 (d, J=8 Hz, 1H,CH_(arom.)), 6.87 (d, J=8 Hz, 1H, CH_(arom.)), 4.37 (m, 2H, OCH₂),3.77-3.40 (m, 10H, CH₂N), 2.14 (s, 3H, CH₃).

General Procedure E (for the Synthesis of Compounds 26-28) Compound 26

As another non-limiting example, 3,4-dichlorophenylisocyanate (250 mg,1.330 mmol) and 2-amino-3-hydroxypyridine (146 mg, 1.330 mmol) weredissolved in 10 mL of anhydrous dioxane. The reaction mixture was warmedto 55° C., stirred under nitrogen over night and then cooled to roomtemperature. The crude was purified twice by crystallization in EtOH toafford 26 (178 mg, 45%) as a white powder.

Compound 27

As another non-limiting example, 3,4-dichlorophenylisocyanate (250 mg,1.330 mmol) and 4-amino-6-methoxypyrimidine (166 mg, 1.330 mmol) wereused following the general procedure E to synthesize 27. At the end ofthe reaction, the crude was dissolved in acetic acid and purified byHPLC (70 to 100% of acetonitrile in water) to afford 27 (224 mg, 54%) asa white powder.

Compound 28

As another non-limiting example, 3,4-dichlorophenylisocyanate (250 mg,1.330 mmol) and 2-amino-4-hydroxy-6-methylpyrimidine (166 mg, 1.330mmol) were used following the general procedure E to synthesize 28.Unfortunately, at the end of the reaction the crude wasn't purified asit wasn't soluble in any solvent tested.

TABLE 2 ATF-4 assays in CRL-2351 cell line. ATF-4 C_(max) C_([5])Structure A_(max) ^(a) (μM)^(b) (μM)^(c) 1

11 5 2 2

11 2.4 1.5 3

11 2.5 1.5 4

13 20 11 5

11 10 6 6

13 5 3 7

NA NA NA 8

8 10 3 9

12 20 <2.5 11

7.5 20 8 12

7 10 7 13

12.5 10 5 15

5.5 10 9 16

7 10 7 17

9 10 3 23

7.5 10 7 24

6 10 7 25

10 10 6 26

12 40 13 27

6.5 40 10 28

5 40 40 ^(a)Maximal activity corresponding to the ration of firefly overrenilla luceferase expression. ^(b)Concentration (in μM) correspondingto the maximal activity. ^(c)Concentration (in μM) corresponding to anactivity threshold of 5.

TABLE 3 Determination of the IC₅₀ (μM) of the compounds by SRB assay.IC₅₀ (μM) Compound 2351 2813 KLN  1 2.6 0.8 >20  2 2.8 1.7 ND  3 0.6 0.13.9  4 9 1.8 >20  5 9.3 1.6 20  6 3.2 1 13.5  7 11 >20 >20  8 2.5 3 17.2 9 2.2 0.7 20 11 5.8 5.3 13.5 12 2.5 2.8 12.3 13 2.9 ND 12.4 15 2.4 ND20 16 2.7 0.9 >20 17 2.3 0.8 18.6 23 4.5 5.1 >20 24 2 3.5 >20 25 5.2 3.1ND 26 2 1.8 >20 27 1.2 0.8 12.5 28 0.7 0.7 15

TABLE 4 In vitro Structure Activity Relationship ofN,N′-Diarylthioureas. Growth Ternary Ternary Inhibition Complex*Complex* IC₅₀** Structure Code 10 μm 3 μm (μm)

1806 3.5 2.5 0.9

1814 2 1.5 2

1815 1 1 >20

1816 2.5 1 1.5

1817 6 1 1.4

1817 7 1 1.4

1819 6 1 1.5

1802 7 1.5 3.5

1836 1 1 2

1803 12 1 1.7

1842 1 1 NA

1778 8 5 0.4

1797 1 1 1.2

1798 5 3 0.4

1820 11 1.5 1.5

1821 1 1 4

1831 1.8 1 NA

1805 8 1.4 1.6

1799 4 4 0.7

1800 4 3 0.5

1801 3 3 0.8

1804 25 6 0.1 NA: Not active; *Fold over basal; **Concentration ofcompound that inhibits proliferation of CRL-2351 human breast cancercells by 50%.

TABLE 5 NSC Activity Activity Activity #* CAS_RN* 10 uM 5 uM 2.5 uM 4953056-73-3 3.74 2.32 774 5394-77-4 22.38 10.06 864 959-36-4 3.57 1.811337 4567-99-1 7.15 1626 5346-51-0 5.31 0.79 1874 613-63-8 3.99 4.278275 22303-26-0 0.81 11235 2390-54-7 9.2 4.88 12134 8.83 12695 5410-88-86.31 12741 5425-32-1 8.35 12750 5450-50-0 4.9 1.79 13268 5423-92-7 3.181.46 14209 5429-71-0 14.32 0.85 14238 5429-90-3 3.02 1.37 14739 4.061.12 14755 6.63 0.68 19143 5444-68-8 3.2 1.72 19763 6957-97-7 13.29 4.6321293 3.16 21533 5436-20-4 8.88 2.03 21620 2152-70-7 16.28 0.74 247943.37 0.55 24863 2083-09-2 3.22 1.87 26101 7770-76-5 4.13 0.78 266727375-42-0 57.61 1.44 26847 8.15 1.83 29215 68-76-8 36.4 30759 6.9 0.8832929 5636-91-9 5.76 0.35 32994 7511-84-4 6.52 2.83 32999 6.87 2.4135424 3.07 2.55 35730 6.76 5.35 36684 6267-09-0 4.2 0.76 38062 6337-31-15.58 2.61 39901 4.1 1.41 42108 6935-42-8 3.7 42112 135-64-8 3.76 3.7643683 550-15-2 5.26 3.73 45171 6300-60-3 3.08 45226 2691-81-8 7.27 3.0345884 27031-73-8 16.23 0.47 47145 6330-21-8 5.16 3.4 49546 4.62 3 5091539077-64-0 7.81 1.11 51522 10.99 1.66 52531 7355-32-0 5.18 0.8 654296827-10-7 3.49 66326 14354-56-4 5.2 66379 2083-55-8 7.36 0.74 68751 4.4676068 900-95-8 3.68 1.02 78577 3.56 0.74 83335 3743-99-5 3.31 2.77 864891027-40-3 3.17 2.73 86537 7.51 3.85 87240 7375-42-0 37.28 1.45 8769516208-00-7 4.42 3.28 88655 19320-23-1 18.32 0.7 89160 9.22 9029922397-40-6 3.21 2.1 90467 3.88 2.6 91815 20329-54-8 15.17 3.3 9181620329-55-9 29.75 17.64 92938 7600-14-8 4.68 3.41 95832 3.2 0.46 969422562-90-5 7.54 4.17 97372 29676-50-4 4.98 1.62 97681 88893-89-4 8.981.17 99639 17958-62-2 8.32 0.7 100622 3.12 2.25 102003 9.46 0.42 10251613266-07-4 3.4 103749 30117-68-1 5.25 2.76 105326 92967-81-2 9.56 1062913.15 2.7 106378 3.69 2.55 106656 35694-47-4 3.26 1.89 107229 3.14 1.06108310 20691-83-2 4.88 3.62 109326 23159-13-9 17.89 0.69 11292161446-06-8 242.82 97.42 113085 1630-53-1 5.05 3.18 114995 20852-34-03.43 2.69 118028 17154-51-7 4.1 119675 8.78 5.52 122844 13.27 1.77122854 13864-38-5 3.2 124738 3.7 0.79 125369 6683-64-3 9.98 1.08 12551613.57 0.79 126650 77800-85-2 8.04 0.64 128665 22242-98-4 3.18 17.83130140 19.67 0.74 130216 92295-16-4 4.77 0.51 131237 27117-05-1 3.3 0.87131464 20958-78-5 3.99 3.63 131584 18754-93-3 7.95 0.89 13166322242-87-1 8.18 7 131747 21628-67-1 3.25 2.54 134404 12.72 5.44 1351557.19 0.94 135854 28558-65-8 5.75 3.56 141112 3.76 0.98 141226 3.5 1.053.8 1.02 142606 34.1 16.57 146184 2381-85-3 3.13 146216 21771-92-6 5.042.73 146249 15672-98-7 3.04 3.25 2.57 149109 59094-49-4 18.18 150279 3.90.62 150781 19802-61-0 3.78 150787 5.6 2.94 151117 12.07 0.85 1511196.62 0.72 155015 73163-54-9 3.73 2.19 157236 3.4 0.75 157306 3.94 1.77157382 23886-57-9 9.04 0.68 157487 34749-63-8 5.81 2.53 157507 3.33 2.07158091 30710-21-5 4.83 1.66 158959 4.01 3.2 161089 19161-98-9 3.53 1.67162375 3.07 1.7 163547 40114-82-7 4.38 3.27 163948 102-98-7 5.67 1642124.07 2.04 165765 60696-76-6 4.44 1.34 168745 15963-72-1 36.37 0.39170334 4.45 0.98 170444 12.56 0.66 170582 24596-38-1 4.44 3.72 17058917010-61-6 6.57 3.81 170600 63019-82-9 3.46 2.21 170633 17076-37-8 8.995.12 170639 92296-17-8 5.38 3.93 171129 7.98 5.1 171134 3.68 2.89 1728793.6 1.11 173000 3.68 0.61 173710 57013-87-3 16.35 0.83 174877 3.29 3.04176145 108-07-6 3.81 176657 4.64 1.67 179739 27605-35-2 3.62 0.98 17976218.35 0.72 179764 25094-60-4 179944 52494-54-9 6.1 0.54 187755 3.08 1.12193484 22.72 1.24 193713 52197-19-0 24.26 0.55 194646 6.24 8.08 19480777547-08-1 3.45 1.72 201570 3.02 2.16 202491 18723-92-7 3.05 0.5 20267421227-23-6 4.67 203205 994-71-8 3.12 203373 20745-97-5 5.25 10.51 20342320745-98-6 3.09 7.7 204163 56661-50-8 4.83 2.44 204246 3.49 1.92 2045123837-55-6 4.16 2.67 204514 3010-57-9 4.66 3.6 204515 3789-77-3 5.31 2.84204548 3.99 2.82 204716 37666-22-1 3.01 2.2 204750 29771-69-5 24.26 3.53204751 4.76 2.91 205530 73190-69-9 3.34 3.08 205545 4.54 2.41 2055484.01 2.94 205787 4.39 1.79 205789 7.26 3.99 212379 31191-41-0 4.4 2.28229344 4.4 1.28 234486 10 236237 57.44 1.78 240577 4.63 2.68 258618 3.20.85 260514 3.87 1.94 262421 76609-51-3 107.79 24.56 263517 3529-04-28.78 1.39 268393 70380-40-4 0.71 270151 3.16 1.24 271272 71007-82-4 4.932.76 271654 18.07 0.44 276293 40919-31-1 20.35 278352 3.81 280894 2.06281986 3561-04-4 3.37 3.11 286678 1124-50-1 1.72 287407 36.2 3.93 28741317.06 0.68 291104 83379-23-1 3.83 1.4 293939 36539-81-8 7.78 1.54 2954779.07 6.24 295679 75082-14-3 3.69 0.78 297153 67675-62-1 4.39 0.95 29715467675-64-3 5.62 1.24 297156 5.15 1.08 297621 59404-25-0 18.95 29815364862-32-4 3.43 3.67 299107 279.99 8.13 300554 56213-52-6 8.96 0.48300559 4.71 0.71 306697 19.5 1.77 309898 13.68 1.07 310008 4256-25-14.92 1.66 310545 6.96 0.46 313162 4.03 1.07 315819 67507-09-9 13.17 2.12315820 17.6 1.5 316746 6.11 0.88 319697 75602-17-4 3.1 319699 75602-13-03.62 0.29 322306 63.6 7.11 323938 5.53 0.99 324978 9.9 6.13 32605982641-26-7 50.81 7.62 326123 77921-36-9 12.9 0.47 332426 71508-74-2 3.152.37 337856 25.44 5.52 338631 3.39 1.12 339119 71568-54-2 4.42 1.01339567 39997-88-1 11.94 1.94 340215 3.11 2.4 340302 36536-22-8 4.54 3.29343387 3.33 2.32 344506 71781-96-9 5.08 0.69 349125 13.75 0.47 3532557487-94-7 3.3 353738 7.02 354087 10.14 2.65 354215 66646-01-3 4.14 3.08355167 79441-89-7 4.17 3.09 359827 6.27 0.61 360648 3.85 1.88 361407 3.70.62 361597 13969-30-7 17.56 0.32 361889 91327-55-8 12.01 0.88 36623388061-94-3 30.55 17.26 367620 16.04 0.49 369110 73723-79-2 6.46 1.84369741 84405-20-9 6.34 0.96 370463 52837-61-3 5.98 4.76 370589 39.5411.87 374924 6.35 2.1 374999 15.92 9.15 376265 7.26 2.72 376674 16 9.42377376 5.14 0.96 377377 34.93 0.77 377378 87405-03-6 21.49 0.87 37783123047-95-2 3.79 2.19 378135 3.15 1.75 379578 27.15 0.61 37966567485-29-4 35.03 1.08 380207 88000-87-7 6.39 0.47 400077 888-71-1 3.142.04 400538 2150-58-5 6.8 0.57 400939 7467-29-0 3.56 2.36 4012347621-92-3 24.65 1 401304 7469-11-6 43.11 1.04 402193 1940-43-8 3.19 0.56402291 4.43 0.73 402592 2703-27-7 3.48 2.25 402600 4638-48-6 4.82 1.14402785 50.88 402826 5.42 0.85 402866 26.02 1.66 403440 7404-15-1 3.831.41 403488 7502-07-0 4.41 1.55 403534 9.9 7.33 405522 6043-40-9 3.692.47 406018 7509-97-9 4.09 1.51 406824 7497-80-5 4.25 1.76 407396426-79-9 4.74 1.72 407658 7499-45-8 5.17 1.57 408380 4.36 2.28 40838310.77 0.84 408399 3.42 1.63 408711 21.88 1.47 408712 21.65 1.57 40977719434-42-5 5.55 20.85 601076 3.17 2.48 601994 5.21 3.42 602807 3.12 2.08603554 8.45 0.35 603854 4.19 0.93 604861 4.82 0.8 604868 5.36 0.86607085 3.55 2.21 608144 112.47 3.82 609211 18.74 5.68 609810 4.69 2.44610051 9.23 1.52 615538 4.53 1.65 617570 94.55 2.36 617738 7.65 1.05617743 3.08 1.47 618767 6.58 0.7 620291 9.07 8.39 620296 3.13 5.24620852 16.94 4.79 621482 5.15 1.17 621504 14.68 621792 3.75 1.27 6217946.6 9.24 621795 18.49 621796 26.11 621797 30.28 25.08 621882 3.1 2.37622579 6.29 2.99 622683 3.82 2.84 623141 4.84 0.53 624851 12.65 6258633.72 3.75 627459 7.21 1.68 628577 3.06 1.45 628578 3.53 628594 3.94631943 6.31 631945 5.72 0.76 631946 13.13 632134 3.5 1.26 633123 3.880.89 633268 16.53 3.96 633334 28.14 4.12 633346 11.64 8.05 633992 13.09633995 11.56 14.38 633997 12.56 17.11 634000 12.96 1.1 634004 9.64 3.7634014 12.92 11.17 634157 3.99 0.8 634838 30.9 13.01 634842 8.37 1.71635009 10.57 5.71 635022 4.8 0.96 635030 71.33 1.68 635072 5.58 1.59638113 3.17 1.87 642485 3.59 1.93 643139 1.53 643713 4.87 2.72 6437623.47 1.99 644907 4.97 645170 5.1 3 647131 17.42 1.96 647134 3.1 6475903.22 2.31 648479 3.37 2.55 650027 4.42 4.2 652047 3.51 1.15 652257 8.743.09 652531 12.9 0.71 652594 14.89 1.46 652890 7.57 2.23 652917 3.312.33 652924 3.58 1.78 7.29 655141 3.91 2.18 656075 7.97 2.33 65607617.27 4.6 656711 6.66 0.59 657189 3.16 2.65 657424 3.36 1.17 658163 4.462.63 658355 8.48 0.5 658358 20.35 0.4 658830 4.21 0.48 659608 3.07 1.34661223 11.8 1.05 661225 25.16 1.76 662875 40.72 10.55 664259 4.24 2.76665512 15.46 0.47 665702 3.38 1.79 665910 4.69 0.72 666131 8.72 4.12666356 30.26 2.43 667223 6.01 0.42 667226 4.79 0.34 667463 3.45 3.13667472 9.48 0.35 667875 7.27 4.59 667885 15.62 8.61 668262 3.33 0.78668297 37.53 10.51 668298 30.06 11.99 668332 63.38 27.24 668484 3.571.59 668494 13.68 3.76 668605 3.17 0.8 670779 6.67 2.57 670788 4.08 6.58670961 4.46 7.92 670965 23.32 50.7 671441 5.35 671442 7.03 9.59 6718964.31 0.85 673797 4.7 2.54 674469 23.82 1.37 678036 12.77 0.85 6808344.25 0.76 682512 10.02 0.57 687849 12.02 0.88 690404 3.03 1.46 69073410.02 0.48 186257 52197-26-9 59.966 49.982 185066 20.339 44.91067 11802617154-55-1 40.338 37.512 150311 39.7 36.66967 75382 15091-30-2 21.05533.125 273747 64724-84-1 26.646 29.296 174794 52197-13-4 33.869 24.857150320 16.4 19.9 165688 22933-76-2 14.6965 17.91033 94582 17.45817.82567 65486 3820-71-1 20.8755 17.496 177407 58885-11-3 21.876 16.53135331 31.606 15.388 99047 17490-47-0 13.186 14.82433 148342 19.755514.51333 101539 6.8175 14.033 327371 71156-12-2 22.247 13.29167 558696947-89-3 13.613 13.23667 83318 88210-37-1 14.6225 12.02733 8800121.4655 12.00033 58338 6627-15-2 17.599 11.604 54905 16.011 11.55767167334 38633-42-0 12.373 11.387 72005 101-20-2 16.9235 11.24533 3433858.3265 11.19733 93360 485-72-3 10.283 11.07867 98409 10432-50-5 25.65910.997 152111 16436-29-6 10.391 10.85133 79681 5.9 10.5 83089 2428-35-55.1265 10.155 209832 4.7795 9.509333 89864 10.1 9.4 133463 1166-52-54.63 8.8 135741 25201-67-6 7.226 8.604667 321198 9.062 8.313333 731184273-92-1 10.008 8.006333 156572 7.9345 7.884 82910 2785-54-8 12.44257.653667 81523 8.879 7.525667 328479 2867-96-1 7.342 7.501667 68292 18.37.2 115157 40.7 7.1 135744 32251-73-3 21.9 7 693037 8.088 6.794333100239 54980-33-5 7.6705 6.748 214041 8.374 6.705333 276393 64724-83-08.706 6.644333 87086 5.461 6.572333 90777 57532-86-2 8.406 6.553 1044988.4845 6.367333 240870 27128-58-1 7.2395 6.351667 63346 21970-53-66.8235 6.234333 240575 7.0215 6.229667 59070 7.423 6.227 33775722295-55-2 5.978 6.204667 69625 13208-31-6 4.8565 6.200667 1091567204-43-5 9.1 6.2 367416 74396-45-5 7.3545 6.047333 227290 61471-39-46.8065 5.934667 268776 5.5035 5.767 71689 7.139 5.723667 69510 10.9 5.674740 7533-73-5 4.1 5.6 326181 67829-21-4 7.027 5.584333 240576 6.28855.469667 57019 2872-52-8 5.441 5.388 204668 4.923 5.386 34621272499-61-7 5.7595 5.376 214004 6.0655 5.299333 87323 30041-69-1 6.09055.277333 204386 6.0225 5.251667 214029 6.8665 5.244667 104959 6824-07-35.474 5.189667 369061 93745-54-1 6.3 5.136667 321197 5.0845 5.014333694092 5.898 4.958 687753 6.931 4.921667 86430 2428-30-0 6.063 4.871333689962 7.987 4.784333 101082 5784-95-2 5.896 4.772333 292796 52053-74-45.6 4.759333 136768 28069-65-0 3.271 4.667667 213848 3.5355 4.586 6912565.7865 4.563 338462 7560-35-2 6.4125 4.540333 222612 2.88 4.521333186948 4.4425 4.507333 230359 72648-43-2 8.3955 4.464667 76429 5.24054.409333 356111 90760-42-2 3.2 4.4 159209 612-81-7 8.5 4.3 5489574305-79-6 4.975 4.287667 332423 71536-10-2 5.4265 4.287 34720472499-57-1 6.6455 4.245667 343386 5.4775 4.235333 55240 6951-36-6 5.24.2 213710 5.091 4.183 78949 7.7 4.1 231980 31392-73-1 3.8 4.1 33254388324-30-5 5.182 4.082667 119026 90111-22-1 12.6 4 115724 21299-50-3 7.34 691255 5.5705 3.963 86544 637-47-8 4.845 3.953667 136889 16.7 3.9109535 6.3 3.9 691258 3.904 3.891333 290436 53655-17-7 20.4 3.8 3274146.977 3.793333 91885 2440-22-4 4.473 3.742333 191390 73108-79-9 4.2953.709667 292795 35299-76-4 4.66 3.69 202060 64985-95-1 5.049 3.629667191395 42174-34-5 7.0185 3.608667 240724 4.257 3.594 69580 6959-97-3 6.43.5 149581 41962-27-0 4.3 3.5 338119 6443-79-4 5.6885 3.434667 552376624-17-5 6.6 3.4 56345 3.64 3.4 82278 3.1525 3.287667 72035 87-22-93.8265 3.275 186256 52197-25-8 3.71 3.212333 159569 5.804 3.210333135745 32251-74-4 6.5 3.2 122241 20286-82-2 5 3.2 94585 3.525 3.148333347816 72499-65-1 3.5245 3.142667 85646 4.18 3.126667 75971 3.33553.111333 204931 93008-58-3 3.539 3.100333 67112 16.3 3.1 10634461653-37-0 4.1 3.1 103755 16504-14-6 3.394 3.089667 99696 10499-10-23.4275 3.031333 191346 4.4055 3.017667 61369 5137-55-3 4.4 3 722667149-62-4 3.233 2.946333 71676 786-50-5 4.1015 2.921667 71690 34243-33-93.4315 2.888667 112668 13228-40-5 3.3905 2.854333 61626 2508-13-6 3.16852.793 101544 16722-41-1 3.1185 2.767 298243 64273-27-4 3.327 2.747170680 1123-51-9 6.8 2.7 93861 3.171 2.622 71968 7779-17-1 3.5615 2.615161504 22974-38-5 5.3 2.6 106343 5302-41-0 4.1 2.6 103842 27702-26-7 3.32.6 292587 239-58-7 3.294 2.576667 343979 3.1255 2.571333 36933184859-31-4 2.7605 2.533 136886 28.5 2.5 230415 2829-28-9 3.3005 2.468333119688 3.9 2.45 123507 3568-90-9 10.5 2.41 366583 3.134 2.409 35611084633-99-8 3.9 2.4 101484 19749-34-9 3.4 2.4 120440 30251-61-7 3.8 2.3168465 50286-86-7 6 2.2 96306 5.7 2.2 163454 10173-53-2 3.5 2.2 1392215064-89-1 9 2.1 74568 4.8 2.1 85573 3.1 2.1 96375 5.9 2 94029 5659-13-23.2135 1.933667 116685 13432-87-6 4.5 1.9 59465 7400-23-9 6.8 1.8 10620874037-43-7 4.3 1.8 146007 3.4 1.8 55879 6947-93-9 3.8 1.7 7456613595-34-1 3.7 1.7 172656 53219-25-3 3.9 1.6 208394 13.4 1.5 112369 3.11.4 144126 7.3 1.3 106909 22966-82-1 3.4 1.2 55867 6947-88-2 3.2 1.111235 2390-54-7 27.11026 7.299852 163482 3.207313 1.587711 38020788000-87-7 3.061186 0.36152 401162 64693-19-2 7.084166 0.378361 5273477385-99-1 5.590928 2.550671 622579 10.08266 0.38273 643139 7.5111980.181398 653438 12.31648 0.754953 658354 6.608676 0.23793 6589163.180133 2.046552 667223 8.726255 0.154498 670779 8.739112 2.72082670961 14.2083 3.272683 670965 25.3088 44.32161 670969 3.643563 1.549795*Entering either the NSC number (NSC followed by the number) of acompound or the CAS number of the compound in the PubChem compounddatabase will bring up the chemical structure and other publiclyavailable information about the compound.

TABLE 6 Effect of anti-cancer agents (20 μM) on F-luc/ R-luc ratio inthe ternary complex assay. Relative Mechanism F-luc/R-luc Agent ofAction Ratio (+/− SEM) Camptothecin Topoisomerase inhibitor 1.3 + 0.3Colchicine Inhibitor of tubulin polymerization 1.2 + 0.3 Threo-1-phenylGlucolipid synthase inhibitor 1.5 + 0.4 Mitomycin C Alkylating agent,DNA 0.9 + 0.3 synthesis inhibitor H-89 PK-A inhibitor 1.5 + 0.65-fluorouracil Thymidilate synthase inhibitor   1 + 0.2 EpigallocatechinLaminin Receptor 1 activation 3-isobutyl-1- Phoshodiesterase inhibitor1.1 + 0.2 methylxanthine Dilthiazem Ca++ channel blocker 1.4 + 0.4Amiloride Na+ channel blocker 1.1 + 0.3 Okadaic acid Protein Phoshatase1 inhibitor   1 + 0.3 Somatostatin Inhibitor of growth hormone 0.9 + 0.2secretion Glycyl-1-histidyl Not known   1 + 0.1 acetate EtopositeTopoisomerase I inhibitor   1 + 0.2 CLT Ca⁺⁺ store depletion   6 + 1.1

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing description is provided forclarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above examples, but areencompassed by the following claims. All publications and patentapplications cited above are incorporated by reference herein in theirentirety for all purposes to the same extent as if each individualpublication or patent application was specifically indicated to be soincorporated by reference.

What is claimed is:
 1. A compound of formula